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Thermo Scientific Pierce Protein Purification Technical ...
Transcript of Thermo Scientific Pierce Protein Purification Technical ...
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Thermo Scientific Pierce Protein Purification Technical Handbook
Version 2
Introduction for Protein Purification 1-5
Purification Accessories – Protein Extraction, Binding and Elution Buffers 6-11
Protease and Phosphatase Inhibitors 8 for Protein Purification Buffers for Protein Purification 9Spin Cups and Columns 10Disposable Plastic and Centrifuge Columns 11
Fusion Protein Purification 12-21
His-Tagged Protein Purification Resin 14-15Cobalt Resin, Spin Columns and 16-17 Chromatography Cartridges High-quality purification of GST-fusion proteins 18-19GST- and PolyHis-Tagged Pull-Down Assay Kits 20-21
Covalent Coupling of Affinity Ligands to Chromatography Supports 22-35
Covalent Immobilization of Ligands 22-25Products for Immobilizing Ligands 26-29 through Primary Amines Products for Immobilizing Ligands 30-32 through Sulfhydryl Groups Products for Immobilizing Ligands 33 through Carbonyl Groups Products for Immobilizing Ligands 34-35 through Carboxyl Groups
IP/Co-IP 36-45
Immunoprecipitation 36 Traditional Methods vs. 37 Thermo Scientific Pierce Innovations for Co-IP Approaches to Co-IP Free of Antibody Interference 37Optimization Parameters in IP and Co-IP 38Evaluating a Co-IP-Captured Interaction 38IP and Co-IP Kits 39-45
Protein Enrichment 46-57
Phosphoprotein Enrichment Kits 46-47 SH2 Domain Phosphotyrosine Capture Kits 48-50 Use of Titanium Dioxide for 51-52 Phosphopeptide Enrichment Pierce Fe-NTA Phosphopeptide Enrichment Kit 53-54Cell Surface Protein Isolation Kit 55Glycoprotein Isolation Kits 56Ubiquitin Enrichment Kit 57
Antibody Purification 58-67
Overview 58 Immobilized Protein L, Protein A, Protein G 59-61 and Protein A/G IgG Binding and Elution Buffers for 62 Protein A, G, A/G and L Melon Gel Purification Products 63Thiophilic Gel Antibody Purification 64IgM and IgA Purification 65
Avidin:Biotin Binding 66-69
Biotin-binding Proteins 66Immobilized Avidin Products 67Immobilized Streptavidin Products 67Immobilized NeutrAvidin Products 68Immobilized Monomeric Avidin and Kit 68Immobilized Iminobiotin and Biotin 69
FPLC Cartridges 70-77
Overview 70His-tagged Protein FPLC Purification 71-72GST-Tagged Protein FPLC Purification 72Antibody FPLC Purification 73-75Phosphoprotein FPLC Purification 75Biotinylated Protein FPLC Purification 76Protein Desalting 77
Affinity Supports 78-80
Table of Contents
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1
General Protein Purification Techniques
Protein purification is essential for a host of biochemical appli-cations. However, with thousands of proteins each displaying unique characteristics, it is important to develop a strategy for purification that delivers the correct yield, purity and activity needed for downstream applications. Forlowresolution/highyieldproteinpurification,methodssuchasfractionalprecipitationusingsaltssuchasammoniumsulfateexist.Forapplicationsrequiringthehighestpurityandrelativelysmallamountsofprotein,affinitypurificationtechniquescanbechosentoselectivelyextractatargetproteinfromthecomplexmixtureofproteinsfoundincellortissueextracts.Table1summarizesafewgeneralstrategies.
Eachoftheseproteinpurificationtechniquesrequiresspecificbuffers(mobilephase),chromatographyresins(solidphase)andcolumnaccessories.Thesethreecomponentscanbeusedinavarietyofdifferentconfigurations,basedonthescaleofpurifica-tionandavailableequipment.Commonformatsinclude:
Batch purification Mixingthemobileandstationaryphasesinaconicaltubeandseparatingthetwoviacentrifugation.Batchmethodpurificationcanbeperformedatanyscale.However,itismostcommonlyreservedformicrocentrifugetubescalepurificationsinvolving10-200μLofresin.Inbatchmethodpurification,washandelutionfractionsareseparatedfromtheresinaftercentrifugingtopellettheresinbeads.Theliquidcannotberemovedcompletelybecausesomeofitiscontainedwithinthevolumeofporousbeadpellet.Consequently,aportionofeachfractionaboutequaltothevolumeofresinusedisleftbehindinthepellet,makingwashesandelutionsomewhatinefficient.
Gravity flow chromatography Passivelyaddingthemobilephasetopackedcolumns,withoutmechanicallyincreasingtheflowrate.Gravityflowcom-monlyuses1mL-to5mL-packedcolumnsset-uponabenchorinachromatographyrefrigerator.Largercolumnscanbepackedtosupportlargerscaleproteinpurification.However,theweightoftheresininthecolumnmustbeconsideredtopreventdamagetoresinatthebottomofthecolumn.Gravityflowallowsforextendedbinding,washingandelutiontimes,whichisidealforsampleswithlowbindingaffinity.
Introduction to Protein Purification
Table 1. Summary of protein purification techniques.
Resolution/ purity Technique Protein Yield Description
Low
Ammoniumsulfateprecipitation High Fractionalprecipitationofproteinsbasedontheirsolubilityinsaltsolutionsofvaryingsaturation
Hydrophobicinteractionchromatography High Separationofproteinsbasedintheirsurfacehydrophobicity
Sizeexclusionchromatography(SEC) Medium Separationofproteinsbasedonmolecularsize
Medium
Ionexchangechromatography Medium SeparationbasedonproteinchargeataparticularpH
Gelelectrophoresis Medium Two-stepseparationofproteinbasedonsizeusingpolyacrylimidegelelectrophoresis(SDS-PAGE)andchargeusingisoelectricfocusing.Note:Thisisadenaturingmethodthatresultsinacompletelossofproteinactivity.
Proteomefractionation Medium Enrichmentofclassesofproteins,suchas:•phosphoproteinsusingimmobilizedmetalaffinitychromatography(IMAC)•glycoproteinsusingimmobilizedlectins•nitrosylatedorpalmitylatedproteinsusingthebiotinswitchassay•organelle-specificproteomesusingcellularfractionationtechniques
High
Affinitypurification Low Selectivepurificationusingaffinitytagsattachedtothetargetgene,suchaspolyhistidine(6xHis),glutathioneS-transferase(GST)ormaltosebindingprotein(MBP)
Immunoprecipitation Lowest Antibody-basedextractionofasingleproteinspecies,typicallyfromcellortissuelysatesh
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Spin cup purificationSeparatingthemobileandstationaryphasesusingcentrifugationinpackedspintubeswithfiltersthatretainresinincolumn.Spinpurificationcanalsobeperformedwithspinplates,whereeachwellofa96-wellmicrotiterplatehasafilterbaseandispackedwiththeappropriatechromotagraphyresin.Thespincuppurificationmethodprovidesimprovedefficiencyofwashandelutionstepscomparedtothebatchmethod.Centrifugationseparatestheliquidfractionbypullingitthoroughlyfromtheresin,whichisretainedwithinthespincupapparatus.Spincuppurifica-tionismostappropriatewhen50-300μLofimmobilizedligandresinisused.
Magnetic purificationIsolatingthestationaryphaseusingmag-nets.Magneticbeadsarecommonlyironoxideparticleswhicharecoatedwithaligandtopurifyaproteintarget.Anexampleofthisisamagneticparticlecoatedwithreducedglutathione(GSH)usedtopurifyGST-taggedrecombinantproteins(Product#88821).Advantagesofusingmagneticbeadsincludeeasyhandling,minimallossofresinfrompipettingandcompatibilitywithhighthroughoutautomatedsystemssuchastheThermoScientificKingFisher96instrument.
Fast protein liquid chromotagraphy (FPLC)Usingchromatographycartridgespre-packedwiththestationaryphaseandaseriesofpumpsandUVdetectorstomoveandmonitorthemobilephase.TheadvantagesofusingFPLCincludereducedpurificationtimesandtheabilitytoimproveresolutionbylinkingmultiplecolumnsintandemforgreaterseparation.
Affinity Purification
Variousmethodsareusedtoenrichorpurifyatargetproteinfromotherproteinsandcomponentsinacrudecelllysateorothersample.Themostpowerfulofthesemethodsisaffinitypurification,alsocalledaffinitychromatography,wherebytheproteinofinterestispurifiedusingitsspecificbindingpropertiestoanimmobilizedligand.
Affinitypurificationmakesuseofspecificbindingthatoccursbetweenmoleculesandisusedextensivelyfortheisolationofbiologicalmolecules.Asinglepassthroughanaffinitycolumncanachievea1,000-to10,000-foldpurificationofaligandfromacrudemixture.Fromasingleaffinitypurificationstep,itispossibletoisolateacompoundinaformpureenoughtoobtainasinglebanduponSDS-PAGEanalysis.Weofferanumberofimmobilizedproteinorligandproductsforaffinitypurificationofantibodies,fusion-taggedproteins,biotinylatedproteinsandotherproteinsforwhichanaffinityligandisavailable.
Inaffinitypurification,aligandisimmobilizedtoasolidsupport.Onceimmobilized,itspecificallybindsitspartnerundermildbufferconditions(oftenphysiologicalconditionssuchasphosphatebufferedsaline).Afterbindingtothepartnermolecule,thesupportiswashedwithadditionalbuffertoremoveunboundcomponentsofthesample.Anelutionbufferisadded,disruptingtheinteractionbetweentheligandanditsbindingpartnerbypHextremes(loworhigh),highsalt,detergents,chaotrophicagentsortheremovalofsomefactorrequiredforthepairtobind.Oncereleased,thebindingpartnercanberecoveredfromthesupportusingadditionalelutionbuffer.Theelutionbuffercanthenbeexchangedbydialysisordesaltingintoamoresuitablebufferforstorageordownstreamanalysis.
Activatedaffinitysupportproductsandkitsenablearesearchertoimmobilizenearlyanytypeofligandtopurifyitsbindingpartner(s).Forexample,ifapeptideantigenisusedtoimmunizeanimalsandproduceantibodies,thesamepeptidecanbeimmobilizedtoagelsupportandusedtoaffinity-purifythespecificantibodyfromanimalserum.Alternatively,ifaspecificantibodyisavailableagainstaparticularproteinofinterest,itcanbeimmobilizedtoasupportandusedtoaffinity-purifytheproteinfromcrudecelllysate.Purificationwithrespecttonearlyanybindinginteractioncanbemadebythisapproach.
Introduction to Protein Purification
3To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.
Affinitypurificationproductsusingeitherimmobilizedligandsoractivatedaffinitysupportchemistriesareavailableforuseinseveraldifferentformats.Mostcommonly,porousbeadedgelsupportsareusedforgravity-column,spin-columnorbatch-scalepurificationprocedures.Coatedmicroplatesareavailableforhigh-throughputscreeningapplications,andmagneticparticlesareespeciallyusefulforautomatedproteinpurification.
Proteinsandothermacromoleculesofinterestcanbepurifiedfromcrudeextractsorothercomplexmixturesusingavarietyofmethods.Precipitationisperhapsthesimplestmethodforseparatingonetypeofmacromoleculefromanother.Forexample,nucleicacidscanbeprecipitatedandtherebypurifiedfromundesiredmoleculesinsolutionusingethanol,andproteinscanbeselectivelyprecipitatedinthepresenceofammoniumsulfate.
Mostpurificationmethodsinvolvesomeformofchromatographywherebymoleculesinsolution(mobilephase)areseparatedbasedondifferencesinchemicalorphysicalinteractionwithastation-arymaterial(solidphase).Gelfiltration(alsocalleddesalting,sizeexclusionchromatographyorSEC)usesaporousgelmaterialtoseparatemoleculesbasedonsize;largemoleculesareexcludedfromtheinternalspacesofthegelmaterialwhilesmallmoleculesentertheresinpores,resultinginalongerpaththroughthecol-umn.Inionexchangechromatography,moleculesareseparatedaccordingtothestrengthoftheiroverallionicinteractionwithasolid-phasematerial.Bymanipulatingbufferconditions,moleculesofgreaterorlesserioniccharactercanbeboundtoordissociatedfromthesolid-phasematerial.
Incontrast,affinitychromatographyoraffinitypurificationmakesuseofspecificbindinginteractionsbetweenmolecules.Aparticularligandischemicallyimmobilizedor“coupled”toasolidsupportsothatwhenacomplexmixtureispassedoverthecolumn,onlythosemoleculeshavingspecificbindingaffinitytotheligandarepurified.Affinitypurificationgenerallyinvolvesthefollowingsteps:
1.Incubatecrudesamplewiththeimmobilizedligandsupportmaterialtoallowthetargetmoleculeinthesampletobindtotheimmobilizedligand.
2.Washawayunboundsamplecomponentsfromsolidsupport.
3.Elute(dissociateandrecover)thetargetmoleculefromtheimmobilizedligandbyalteringthebufferconditionssothatthebindinginteractionnolongeroccurs.
Ligandsthatbindtogeneralclassesofproteins(e.g.,ProteinAforantibodies)orcommonlyusedfusionproteintags(e.g.,glutathioneforGST-taggedproteins)areavailableinpre-immobilizedformsreadytouseforaffinitypurification.Alternatively,morespecializedligandssuchasspecificantibodiesorantigensofinterestcanbeimmobilizedusingoneofseveralactivatedaffinitysupports;forexample,apeptideantigencanbeimmobilizedtoasupportandusedtopurifyantibodiesthatrecognizethepeptide.
Mostcommonly,ligandsareimmobilizedor“coupled”directlytosolidsupportmaterialbyformationofcovalentchemicalbondsbetweenparticularfunctionalgroupsontheligand(e.g.,primaryamines,sulfhydryls,carboxylicacids,aldehydes)andreactivegroupsonthesupport.However,othercouplingapproachesarealsopossible.IntheThermoScientificGSTOrientationKit(Product#78201),forexample,aGST-taggedfusionproteinisfirstboundtoanimmobilizedglutathionesupportbyaffinityinteractionwiththeGSTtagandthenchemicallycrosslinkedtothesupport.TheimmobilizedGST-taggedfusionproteincanthenbeusedtoaffinity-purifyitsbindingpartner(s).Likewise,theThermoScientificPierceCrosslinkImmunoprecipitationKits(Product#26147)andThermoScientificIgGOrientationKits(Product#44990)involvebindingandsubsequentcrosslinkingofanantibodytoimmobilizedProteinA,A/G.
Historically,researchershaveusedaffinitypurificationprimarilytopurifyindividualmoleculesofinterest.Increasingly,proteomicsresearchfocusesondeterminationofdiseasestates,celldifferentiation,normalphysiologicalfunctionsanddrugdiscoveryinvolvinginteractionandexpressionofmultiplemoleculesratherthanindividualtargets.Consequently,theuseofaffinitymethodshasexpandedtopurificationofnativemolecularcomplexesandformsthebasisforco-immunoprecipitation(co-IP)and“pull-down”assaysinvolvingprotein:proteininteractions.
1. Immobilize the antigen to an appropriate support.
4. Bind anti-antigen antibodies.
5. Wash off unbound antibodies.
6. Elute anti-antigen antibodies.
2. Quench the unreacted sites and wash.
3. Add the antibody solution.
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Typical antibody purification using an immobilized antigen column.
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Affinity Purification Supports
Affinitypurificationinvolvestheseparationofmoleculesinsolution(mobilephase)basedondifferencesinbindinginteractionwithaligandthatisimmobilizedtoastationarymaterial(solidphase).Thesolidphaseinaffinitypurificationisasupportormatrixmaterialthatabiospecificligandmaybecovalentlyattached.Typically,thematerialtobeusedasanaffinitymatrixisinsolubleinthesysteminwhichthetargetmoleculeisfound.Usually,butnotalways,theinsolublematrixisasolid.Hundredsofsubstanceshavebeendescribedandemployedasaffinitymatrices.
Usefulaffinitysupportsarethosewithahighsurfaceareatovolumeratio,chemicalgroupsthatareeasilymodifiedforcovalentattachmentofligands,minimalnonspecificbindingproperties,goodflowcharacteristics,andmechanicalandchemicalstability.Whenchoosinganaffinitysupportormatrixforanyseparation,themostimportantquestiontoansweriswhetherareliablecommercialsourceexistsforthedesiredmatrixmaterialinthequantitiesrequired.Fortunately,weofferawiderangeofpracticalandefficientmatricesinvolumesrangingfrom1mLtomuchlargerbulkquantities.
Porous Beaded ResinsPorousbeadedsupportsgenerallyprovidethemostusefulproper-tiesforaffinitypurificationofproteins.Weofferaffinitypurifica-tionproductsintwomainporousgelsupportformats:crosslinkedbeadedagaroseandThermoScientificUltraLinkBiosupport.Thevariousfeaturesofthesetwosupportsarelistedintheaccompanyingtable.Agaroseisgoodforroutineapplicationsbutcrusheseasily,makingitsuitableforgravity-flowcolumnorsmall-scalebatchproceduresusinglow-speedcentrifugation.UltraLinkBiosupportisincompressibleandcanbeusedinhigh-pressureapplicationswithaperistalticpumporotherliquidchromatogra-physystem.Inaddition,UltraLink®Supportsdisplayextremelylownonspecificbindingcharacteristicsbecausetheyarepolyacryl-amide-based.Bothsupportsperformwellintypicalgravity-flowandspincolumnpurificationandimmunoprecipitationprocedures.
Magnetic ParticlesWhenamatrixisrequiredforaffinitypurificationofcellswithinapopulation,werecommendThermoScientificMagnaBindBeads.Magneticaffinityseparationisaconvenientmethodforisolatingantibodies,antigens,lectins,enzymes,nucleicacidsandcellswhileretainingbiologicalactivity.Samplescontainingthemole-culeofinterestareincubatedwithbeadsthatarederivatizedwithanantibodyorotherbindingpartner.ArareearthmagnetisusedtopulltheMagnaBind™Beadsoutofsolutionandontoasurface.Thebuffercanbecarefullyremoved,containinganynon-boundmoleculesorcells.
MagnaBindBeadsconsistofasilanizedsurfaceoveranironoxidecore(seeTable2).Thesilanizedsurfacehasbeenderiva-tizedtocontainactivegroups,suchascarboxylicacidsorprimaryamines,orspecificaffinitymoleculessuchasstreptavidin;ProteinA;ProteinG;orgoatanti-mouse,anti-rabbitoranti-ratIgG.DuetothenatureoftheMagnaBindBeads,strongelutionconditionsarenotrecommendedwiththeseproducts.Seepage80foracompletelistingofMagnaBindSupports.
Introduction to Protein Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 5
Table 2. Characteristics of underivatized Thermo Scientific MagnaBind Beads.
Composition Silanizedironoxide
Magnetization 25–35EMU/g
Type of Magnetization Superparamagnetic(nomagneticmemory)
Surface Area >100m2/g
Settling Rate 4%in30minutes
Effective Density 2.5g/mL
Number of Beads 1x108beads/mg
pH Stability Aqueoussolution,abovepH4.0
Concentration 5mg/mL
Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with antibiotic medium or g-irradiated.
MicroplatesPolystyrenemicroplatesareanothertypeofmatrixcommonlyusedforimmobilizationofproteins.Proteinspassivelyadsorbtothepolystyrenesurfacethroughhydrophobicinteractions.Generally,thisadsorptionofproteinsontothepolystyrenesurfaceoccursbestincarbonate/bicarbonatebufferatanalkalinepH(9.0–9.5).Inaddition,polystyrenesurfacescanbederivatizedwithcertainchemistriesthatwillallowpeptidesandothernonproteinmoleculestoadheretothesurfacetoperformaffinityassaysinthewellsoftheplates.
Weofferprecoatedplatestoallowresearchersaneasy-to-use,consistentmethodforaffinitypurificationoridentificationofspecificmoleculesofinterest.Theplatesofferedincludethosespecificforfusionproteins(6xHis,GSTandGFP),antibodies(ProteinA,ProteinG,ProteinA/G,ProteinL,goatanti-mouseandgoatanti-rabbitIgG),biotin(streptavidinandThermoScientificNeutrAvidinProtein)andthosewithreactivechemistries(maleicanhydrideandmaleimide)toallowbindingofnonproteinsamplesthatdonotadsorbtotheplasticmicroplatewellsurface.Onlyselectedmicroplateproductsarefeaturedinthishandbook.Foracompleteselectionofprecoatedplates,visitwww.thermosci-entific.com/pierce.
Thereareavarietyofactivatedsupportsthatallowaresearchertopurifyproteinsandotherbiologicalmoleculesofinteresteitheraloneorwhenpresentincomplexeswiththeirbindingpartners.Manyofthesesupportsarediscussedonthefollowingpages.
Physical properties of porous gel supports.
Support
4% Agarose (crosslinked beaded agarose)
6% Agarose(crosslinked beaded agarose)
Thermo Scientific UltraLink Biosupport (co-polymer of crosslinked bis-acrylamide and azlactone)
Bead 45–165μm 45–165μm 50–80μm
Exclusion Limit 20,000,000daltons 4,000,000daltons 2,000,000daltons(1,000Åporesize)
Durability Crushesunderpressure Crushesunderpressure Sturdy(>100psi,6.9bar)*
Types of Chromatography Gravityandsmallspincolumns Gravityandsmallspincolumns FPLCSystems,mediumpressure,gravityflow
Coupling Capacity Medium Medium High
pH range 3–11 3–11 3–11
Form Preswollen Preswollen DryorPreswollen
* Note: The indicated maximum pressure of 100 psi refers to the maximum pressure drop across the gel bed that the support can withstand. It does not necessarily refer to the indicated system pressure shown on a liquid chromatography apparatus because the system pressure may not actually be measuring the pressure drop across the column. Typical system pressures are usually much higher due to
pumping through small I.D. tubing, auto-samplers, detectors, etc. When packed into a 3mm ID x 14cm height glass column, these exclusive supports have been run to approximately 650 psi (system pressure) with no visual compression of the gel or adverse effects on chromatography. These columns can be run at linear flow rates or 85–3,000cm/hour with excellent separation characteristics.
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Protein Extraction, Binding and Elution Buffers
Protein purification is preceeded by expression of the target protein in a host organism. In in vitro protein expression, the simple introduction of DNA and a bacteriophage RNA polymerase to a cell free lysate initiates protein production, with no extrac-tion reagents required before purification. For in vivo protein expression, such as in E. coli or tissue culture cells, expression can be driven from constituitive promoters or it can be induced chemically during bacterial growth or induced genetically through transient DNA transfection into tissue culture cells. For in vivo protein expression, the total protein content of a cell culture or tissue sample must be extracted from the cell's mem-branes and organelles before a chromatography resin can be used for purification. A list of Thermo Scientific Pierce Protein Extraction Reagents for different cell and tissue type is listed on page 7. Additionally, protease and phosphatase inhibitor cocktails that can be used to preserve protein integrity during extraction (see page 8).
Mostaffinitypurificationproceduresinvolvingprotein:ligandinter-actionsusebindingbuffers,suchasphosphatebufferedsaline(PBS),atphysiologicpHandionicstrength.Thisisespeciallytruewhenantibody:antigenornativeprotein:proteininteractionsarethebasisfortheaffinitypurification.Oncethebindinginteractionoccurs,thesupportiswashedwithadditionalbuffertoremoveunboundcomponentsofthesample.
Nonspecific(e.g.,simpleionic)bindinginteractionscanbemini-mizedbymoderateadjustmentstosaltconcentrationorbyaddinglowlevelsofdetergentinthebindingand/orwashbuffer.Finally,elutionbufferisaddedtobreakthebindinginteractionandreleasethetargetmolecule,whichisthencollectedinitspurifiedform.ElutionbuffercandissociatebindingpartnersbyextremesofpH(loworhigh),highsalt(ionicstrength),theuseofdetergentsorchaotropicagentsthatdenatureoneorbothofthemolecules,removalofabindingfactor,orcompetitionwithacounterligand.Inmostcases,subsequentdialysisordesaltingisrequiredtoexchangethepurifiedproteinfromelutionbufferintoamoresuitablebufferforstorageordownstreamanalysis.Formoreinformationondialysisordesalting,downloadorrequestourafreehigh-performancedialysistechnicalhandbook.
Themostwidelyusedelutionbufferforaffinitypurificationofproteinsis0.1Mglycine•HCl,pH2.5–3.0.Thisbuffereffectivelydissociatesmostprotein:proteinandantibody:antigenbindinginteractionswithoutpermanentlyaffectingproteinstructure.However,someantibodiesandproteinsaredamagedbylowpH,soelutedproteinfraction(s)shouldbeneutralizedimmediatelybycollectingthefractionsintubescontaining1/10thvolumeofalkalinebuffersuchas1MTris•HCl,pH8.5.Otherelutionbuffersforaffinitypurificationofproteinsarelistedintheaccompanyingtable.Inaddition,weofferseveralpreformulatedbindingandelutionbuffersdesignedforaffinitypurificationinvolvingantibodies.
Common elution systems for protein affinity purification.
Condition Buffer
pH 100mmglycine•HCl,pH2.5–3.0100mmcitricacid,pH3.050–100mmtriethylamineortriethanolamine,pH11.5150mmammoniumhydroxide,pH10.5
Ionicstrengthand/orchaotropiceffects
3.5–4.0Mmagnesiumchloride,pH7.0in10mmTris5Mlithiumchloridein10mmphosphatebuffer,pH7.22.5Msodiumiodide,pH7.50.2–3.0sodiumthiocyanate
Denaturing 2–6Mguanidine•HCl2–8Murea1%deoxycholate1%SDS
Organic 10%dioxane50%ethyleneglycol,pH8–11.5(alsochaotropic)
Competitor >0.1Mcounterligandoranalog
Protein Purification Accessories
Cell Lysis Technical Handbook
This50-pagehandbookprovidesprotocolsandtechnicalandproductinformationtohelpmaximizeresultsforprotein/geneexpressionstudies.Thehandbookprovideshelpfulhintsandtroubleshootingforcelllysis,proteinpurification,cellfractionation,proteaseinhibitorsandproteinrefolding.(#1601756)
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 7
Thermo Scientific Pierce Protein Extraction Reagents.
Name Description Organisms/Samples
B-PER® - Bacterial Protein Extraction Reagent Product # 90084
Efficient,gentlelysisandextractionofsolubleproteinsfromE. coliandotherbacterialcells.Usesmildnonionicdetergentstodisruptcellsandsolubilizingproteinswithoutdenaturation,eliminatingtheneedforharshmechanicalprocedureslikesonication.
Gram(-)bacteria,S. aureus,H. pylori,E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.
B-PER IIProduct # 78260
SimilartoB-PER,butoptimizedforlowcelldensity,orforproteinswithlowexpressionlevels.
Gram(-)bacteria,S. aureus,H. pylori,E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.
B-PER PBS Product # 78266
SimilartoB-PER,butinPhosphateBuffer.Thisaminefreeformulationisidealforamine-reactivelabelingand/orcrosslinkingapplications.
Gram(-)bacteria,S. aureus,H. pylori,E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.
B-PER with Enzymes Product # 90078
SimilartoB-PER,butkitcontainsDNAseIandlysozyme,whichimprovecellmembraneandDNAdigestionforincreasedyields,andincreasestherecoveryoflargemolecularweightproteinsandinsolubleproteinsfrominclusionbodies.
Gram(-)bacteria,S. aureus, H. pylori, E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.
B-PER Direct with Enzymes Product # 90080
SimilartoB-PERwithEnzymes,butbacteriacanbelyseddirectlyincellculturemedia;idealforscreening96-wellmicroplatesamples.
Gram(-)bacteria,S. aureus, H. pylori, E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.
Y-PER® - Yeast Protein Extraction Reagent Product # 78990
Easy-to-usesolutiongentlydisruptsthetoughyeastcellwallinlessthan20minutesatroomtemperature,usingamilddetergent.Nomechanicaldisruptionneeded;yieldsmorethantwiceasmuchproteinasglassbeadmethods.
S. cerevisiae,Schizo-saccharomyces pombe,C. albicans,B. subtilis,E. coli,P. pastoris,Strep. avidiniiandAcinetobactersp.
Y-PER Plus Product # 78998
MorestringentthanY-PER,butentireformulation(includingdetergent)aredialyzable.
Yeast(S. cerevisiae)andAcinetobactersp.
M-PER® - Mammalian Protein Extraction Reagent Product # 78501
Highlyefficienttotalproteinextractionfromculturedmammaliancells;extractsproteinsinnondenaturedstate,enablingproteintobedirectlyimmunoprecipitated;amine-freeandfullydialyzable;adheredcellscanbedirectlylysedinplateorafterscrapingandwashinginsuspension.
Culturedmammaliancells,COS-7,NIH3T3,Hepa1-6,293,CHO,MDA,MB231andFM2
P-PER - Plant Protein Extraction Reagent Product # 89803
Containsorganiclysingreagentandtwoaqueousreagents,which,inconjuctionwithmildmechanicalagitation,effectivelyextracthighqualityproteinextractsfromplantleaves,stem,root,seedandflowercellswithoutliquidnitrogenorharshmechanicalaids,suchasmortarandpestle.
Multipleplantorgans(leaf,stem,root,seedandflowers);multipleplantspecies(Arabidopsis,tobacco,maize,soybeans,peas,spinach,riceandotherplanttissues);andfresh,frozenanddehydratedplanttissues
T-PER® - Tissue Protein Extraction Reagent Product # 78510
Simple,easytousereagentforextractingtotalproteinfromtissuein1:20(w/v)oftissuetoT-PER,usingcentrifugationtopelletcell/tissuedebris.Milddetergentisdialyzable.
Heart,liver,kidneyandbrain.
I-PER - Insect Cell Protein Extraction Reagent Product # 89802
Optimizedmildnonioinicdetergentformulationprovidesmaximumextractionofsolubleproteinsfrominsectcells;betteryieldthansonication;canbeusedforbothsuspendedoradherentinsectcells
Baculovirus-infectedinsectcellsgrowninsuspensionormonolayerculture.
NE-PER® - Nuclear and Cystoplasmic Extraction Kit Product # 78833
Obtainfunctionalconcentratednuclearextractsandcytoplasmicfractionsfrommammaliancellsandtissuesinlessthantwohours,eliminatingtheneedforfreeze/thawcycles,Douncehomogenization,lengthycentrifugationtimesandcold-roomwork.
Tissue:calfliver.Tissue:mouseheart,kidney,lungandliver;Culturedcells:epithelial(HeLa),fibroid(COS-7),kidney(NIH3T3),liver(Hepa1)andbrain(C6).
Mem-PER® - Eukaryotic Membrane Protein Extraction Kit Product # 89826
Efficient,gentlereagentsthatsolubilizeandisolatemembraneproteinsfrommammalianandyeastcells,aswellassoftandhardtissues,inlessthananhour.Minimalcrosscontamination(lessthan10%)ofhydrophilicproteinsintothehydrophobic(membraneprotein)fraction
Culturedcells:brain(C6),epithelial(HeLa),fibroblasts(NIH3T3)andyeast(S. cerevisiae).
Subcellular Protein Fractionation Kit Product # 78840
Includesacombinationofreagentsforstepwiselysisofmammaliancellsintofunctionalcytoplasmic,membrane,solublenuclear,chromatin-bound,andcytoskeletalproteinfractionsinasinglekit;includesastabilizednucleaseandproteaseinhibitors.Extractsfromeachcompartmenthavelessthan15%contaminationbetweenfractions,withsufficientpurityforstudyingproteinlocalizationandredistribution.
Culturedmammaliancells.
Mitochondrial Isolation Kit for Cultured Cells Product # 89874
Isolateintactmitochondriafromculturedmammaliancellsinapproximately40minutes,withanoptionalDouncehomogenizationprotocolforincreasedyield.
Mammaliancells.
Mitochondrial Isolation Kit for Tissues Product # 89801
Isolateintactmitochondriafromsoftorhardtissueinlessthan60minutes,withanoptionalDouncehomogenizationprotocolforincreasedyield.
Heart,liver,kidneyandbrain.
Lysosome Enrichment Kit for Tissues and Cells Product # 89839
Usesdensitygradientcentrifugationtoseparatelysozymefromcontaminatingcelluarstructuresinbothmammaliancellsandsoftandhardtissue.
Tissuesandculturedcells.
Peroxisome Enrichment Kit for Tissues Product # 89840
Usesdensitygradientcentrifugationtoseparateperoxisomefromcontaminatingcelluarstructuresinbothsoftandhardtissue.
Heart,liver,kidneyandbrain.
Nuclei Enrichment Kit for Tissue Product # 89841
Usesdensitygradientcentrifugationtoseparatenucleifromcontaminatingcelluarstructuresinbothsoftandhardtissue
Heart,liver,kidneyandbrain.
Pierce® RIPA BufferProduct # 89900
Extractscystoplasmic,membrane,andnuclearproteinsfromculturedmammaliancells;canbeusedforbothplatedcellsandcellspelletedfromsuspensioncultures.Proteaseandphosphataseinhibitorsarecompatiblewiththisformulation.
Culturedmammaliancellsandcytoplasmic,membraneandnuclearproteins.
Pierce IP Lysis Buffer Product # 87787
Gentlyextractscytoplasmic,membraneandnuclearproteinswhilemaintainingproteincomplexesforimmunoprecipitation(IP),Pulldowns,andco-IP;doesnotliberateDNAwhichcancausehighviscosity.
Culturedmammaliancells.
For more details on these products, request the Cell Lysis Technical Handbook (#1601756) at www.thermoscientific.com/pierce
8 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Protein Purification Accessories
Protease and Phosphatase Inhibitors for Protein Purification
Thermo Scientific Halt Protease and Phosphase InhibitorsOurbroad-spectrumproteaseandphosphataseinhibitorcocktailsandindividualproteaseinhibitorsaccommodatespecificandgeneralneedsincelllysisandproteinextractionmethods.
Highlights•HaltProteaseInhibitorCocktailstargetserine,cysteineand
asparticacidproteasesandaminopeptidases.MetalloproteasesareinhibitedbytheoptionaladditionofEDTA.Individualproteaseinhibitorstargetingseparateclassesofproteasesarealsoavailableforcustomcocktaildevelopment.
•Halt™PhosphataseInhibitorCocktailscontainchemicalcom-poundsthattargetserine/threonineandtyrosinephosphatases.
•TheHaltProteaseandPhosphataseInhibitorCocktailpreventsproteindegradationandpreservesphosphorylationsimultaneously,providingprotectionthatissimilartotheindividualcocktails.
ForacompletelistingoftheHaltProteaseandPhosphataseInhibitorsatwww.thermoscientific.com.
Buffers for Protein Purification
Thermo Scientific BupH Pack Dry Blend BuffersBupH®Packsarepre-blendedandpre-measureddrymixturesofcommonlyneededbuffersthatareeasytoprepare;simplyemptycontentsoffoilenvelopepackintoabeaker,addultrapurewater,andstirtodissolve.ThepackseliminateweighingtimeandtediouspHadjustments.BupHPackDryBlendBuffersareofferedforuseincommonlaboratorytechniquesandtosupportotherPierceProteinResearchProducts.
Highlights:•Convenient–dissolvecontentsofoneenvelopein500mlofwater andthebufferisreadytouse•Save time and trouble–noweighing,nopHadjustment,noneed
tostockindividualcomponentsandnoneedtomakeandstorelargevolumesofstocksolutioninadvanceofdailyneeds
•Long shelf life–stockingandstorageasdrypackseliminates concernsaboutlong-termstabilityofstocksolutions•Eliminate variables–ourqualitycontrolensuresthateverypack willyieldthesame,consistentbuffer
Ordering Information Product #
Description
Pkg. Size
Applications
Formulation of each pack after reconstitution
U.S. Price
28384 Borate Buffer 40packs Proteinmodificationproceduresthatrequireamine-freebufferatalkalinepH
500mLof50mMborate,pH8.5 $132
28382 Carbonate-Bicarbonate Buffer 40packs MicroplateproteinandantibodycoatingforELISAorRIA
500mLof0.2Mcarbonate-bicarbonate,pH9.4 $125
28388 Citrate-Carbonate Buffer 10packs ProteinimmobilizationtoUltraLink®
Biosupport(Product#53110)100mLof0.6Msodiumcitrate,0.1Msodiumcarbonate,pH9
$ 64
28386 Citrate-MOPS Buffer 10packs ProteinimmobilizationtoUltraLinkBiosupport(Product#53110)
100mLof0.6Msodiumcitrate,0.1MMOPS,pH7.5
$ 58
28390 MES Buffered Saline 10packs Crosslinkingusingcarbodiimide(EDC,Product#22980)
500mLof0.1MMES,0.9%NaCl,pH4.7 $112
28372 Phosphate Buffered Saline (PBS) 40packs Crosslinkingandbiotinylationrequiringamine-freebuffer
500mLof0.1Msodiumphosphate,0.15MNaCl,pH7.2
$135
28374 Modified Dulbecco’s PBS 40packs WashbuffersandantibodydiluentsforELISA,Westernblottingandotherimmunoassays
500mLof8mMsodiumphosphate,2mMpotassiumphosphate,0.14MNaCl,10mMpotassiumchloride,pH7.4
$ 96
2837928376
Tris Buffered Saline 10packs40packs
WashbuffersandantibodydiluentsforELISA,Westernblottingandotherimmunoassays
500mLof25mMTris,0.15MNaCl,pH7.2
$ 61$134
28378 Tris-Glycine-SDS Buffer 40packs RunningbufferforTris-Glycinegelelectrophoresis
500mLof25mMTris,192mMglycine,0.1%SDS,pH8.3
$120
28398 Tris-HEPES-SDS Running Buffer 10 10packs RunningbufferforTris-HEPESelectrophoresiswithPrecise™PrecastGels
500mLof100mMTris,100mMHEPES,3mMSDS,pH8±0.25
$ 36
28380 Tris-Glycine Transfer Buffer 40packs Runningbufferforgeltomembraneelectrophoretictransfer
500mLof25mMTris,192mMglycine,pH8(use20%methanoltodissolve)
$120
Need a larger quantity?
Call our Bulk and Custom department at 1-800-874-3723
or +1 815-968-0747 ext. 300 for pricing and information. Or, visit
www.thermoscientific.com/protein-custom
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 9
Thermo Scientific Concentrated Liquid BuffersSave counter space and time.
Pierce10Xand20XConcentratedBuffersarereadytousewithouthavingtoreconstitutewithultrapurewaterand,inthecaseofTris-GlycineBuffer,methanol.Buffersaredesignedforuseindialysis,cross-linking,enzymeassays,ELISAs,immunohistochem-istry,proteinplate-coating,biotinylationandotherapplications.Keepourconcentratedbufferscloseathandtocoveryourlab’sresearchneeds.
Highlights:•Easy to use–nopacketstoopenandnopowdertodissolve• Increased accuracy–eliminatesthepossibilityofpowder
remaininginapacket• Saves time–10Xor20Xconcentrationeliminatestimespent
waitingforpowdertodissolve• Saves space–storageasconcentratedstockminimizesbench
spaceneededforlargevolumesolutions
PBS Tris-Glycine-SDS Tris-Hepes-SDS
Ordering Information Product #
Description
Pkg. Size
Applications
1X Formulation
U.S. Price
28341 20X Borate Buffer 500mL IdealforproteinmodificationproceduresrequiringaminefreebufferatalkalinePH
500mLof50mMborate,pH8.5 $68
28344 20X Modified Dulbecco'sPBS Buffer
500mL UsedforwashbuffersandantibodydiluentsinapplicationssuchasELISA,Westernblottingandotherimmunoassays
8mMSodiumPhosphate,2mMPotassiumPhosphate,0.14MNaCl,100mMKCl,pH7.4
$68
28346 20X Modified Dulbecco'sPBS Tween-20 Buffer
500mL AwashbufferforELISA,WesternandotherImmunoassaysaswellasablockingbufferforplatebasedassays
8mMSodiumPhosphate,2mMPotassiumPhosphate,0.14MNaCl,100mMKCl,0.05%Tween-20,pH7.4
$68
28348 20X Phosphate Buffered Saline 500mL Itsionicstrengthmakesitidealforcrosslinkingandbiotinylationrequiringaminefreebuffer
0.01MSodiumPhosphate,0.15MNaCl,pH7.5
$68
28352 20X PBS Tween-20 Buffer 500mL AwashbufferforELISA,WesternandotherImmunoassaysaswellasablockingbufferforplatebasedassays
0.01MSodiumPhosphate,0.15MNaCl,0.05%Tween-20,pH7.5
$68
28354 20X TAE Buffer 500mL Historicallythemostcommonbufferusedforagarosegelelectrophoresisintheanalysesofnucleicacids
0.04MTris,0.04MAcetate,0.001MEDTA,pH8.2-8.4
$68
28355 10X TBE Buffer 1L Oftenusedforagarosegelelectrophoresisintheanalysisofnucleicacids
0.089MTris,0.089MBorate,0.002MEDTA,pH8.2-8.4
$65
28358 20X TBS Buffer 500mL UsedforwashbuffersandantibodydiluentsinapplicationssuchasELISA,Westernblottingandotherimmunoassays
25mMTris,0.15MNaCl,pH7.2 $68
28360 20X TBS Tween-20 Buffer 500mL AwashbufferforELISA,WesternandotherImmunoassaysaswellasablockingbufferforplatebasedassays
25mMTris,0.15MNaCl,0.05%Tween-20,pH7.5
$68
28362 20X Tris/HEPES/SDS Buffer 1L ArunningbufferforTris-HEPESelectrophoresiswithPrecisePrecastGels
0.025MTris,0.192MGlycine,0.1%SDS,pH8.5
$45
28363 10X Tris-Glycine Buffer 1L Greatforelectrophoretictransferfromgeltomembrane
0.025MTris,0.192MGlycine,pH8.5 $45
28368 20X Tris/HEPES/SDS Buffer 500mL ArunningbufferforTris-HEPESelectrophoresiswithPrecisePrecastGels
0.1MTris,0.1MHEPES,3mMSDS,pH8+0.25
$68
10 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Spin Cups and Columns
ThermoScientificPierceSpinColumnsareconvenienttoolsformanipulatingsmallvolumesofaffinitysupports(5–500μL)forproteinpurification.Addtheaffinityresinandsampletooneofthecolumns,useamicrocentrifugetoefficientlywashawaycontaminantsandeluteyourpurifiedsamplewithoutlosinganyresinintheprocess.Spincolumnsallowyoutoaffinity-purifymoreproteininlesstime!
Highlights:•Efficientwashingofsamplesmeansfewerwashesareneeded
toremovecontaminatingproteins•Efficientelutionofsamplesmeansmoreantigenand
co-precipitatedproteinsarerecovered•NoresinlossmeansmoreconsistentIPandco-IPresults•NoneedtodecantsupernatantfromIPorco-IPpellet•SpinprotocolsdrasticallyreducethetimerequiredforIPsandco-IPs•Lowprotein-bindingpolypropylenecolumnconstruction
minimizesnonspecificbinding
Spin Cups – Paper FilterPaper filter with collection tubes.
Highlights:•Paperfiltersareresistanttoclogging
fromcellulardebris•ColumnVolume:600μL•ResinVolume:20–300μL•FilterType:Paper,~10μmporesize•CapType:Collectiontubecapfitsontoinsertedspincup
Spin Cups – Cellulose Acetate FilterCellulose acetate filter with collection tubes.
Highlights:•UsedinourIPandCo-IPKits•ColumnVolume:800μL•ResinVolume:20–400μL•FilterType:Celluloseacetate,0.45μmporesize•CapType:Collectiontubecapfitsontoinsertedspincup
Spin Columns – Screw CapScrew cap with Luer-Lok® Adaptors.
Highlights:•Luer-LokAdaptorsallowthesecolumns
tobeusedforsyringe-basedpurifications•ColumnVolume:900μL•ResinVolume:20–400μL•FilterType:Polyethylene,~10μmporesize•Small&largefritoptionsfordifferentsamplesizes•CapType:O-ringscrewtopcaps;press-inbottomplugs
Spin Columns – Snap CapSnap cap with collection tubes.
Highlights:•UsedintheCellSurfaceProteinIsolation
andGlycoproteinIsolationKits•ColumnVolume:1,000μL•ResinVolume:20–500μL•FilterType:Polyethylene,~30μmporesize•CapType:Snapcaponcolumn(nocaponcollectiontube);
press-onbottomcaps
Micro-Spin ColumnsHighlights:•ColumnVolume:400μL•ResinVolume:5–100μL•FilterType:Polyethylene,~30μmporesize•CapType:O-ringscrewtopcaps;press-on
bottomcaps
Ordering Information Product #
Description
Pkg. Size
U.S. Price
69700 Spin Cups – Paper Filter Includescupsandcollectiontubes
50/pkg $109
69715 Microcentrifuge Tubes CollectionTubesforProduct#69700
72/pkg $ 32
69702 Spin Cups – Cellulose Acetate Filter Includescupsandcollectiontubes
50/pkg $108
69720 Microcentrifuge Tubes CollectionTubesforProduct#69702
72/pkg $ 32
69705 Spin Columns – Screw Cap with Luer-Lok Adaptors Includes:SpinColumns,ScrewCapsandColumnPlugs Luer-LokAdaptors LargeFrits(6.8mmdiameter10μmporesize) SmallFrits(2.7mmdiameter10μmporesize) LargeandSmallFritTools
Kit25each5each25each25each1each
$ 99
69725 Spin Columns – Snap Cap with Collection Tubes Includes:SpinColumnsandBottomCaps CollectionTubes
Kit50each100each
$109
89879 Micro-Spin Columns 50/pkg $ 55
Protein Purification Accessories
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 11
Disposable Plastic Columns
Automatic “stop-flow” action provided by porous polyethylene discs prevents column beds from drying out.
Highlights:•Suppliedcompletewithporouspolyethylenediscs,stoppers,endcaps•Compatiblewithmosttypesofaqueousbuffereluentscommonly
usedinchromatography•Canbepre-packedandstoreduntilneeded
Ordering Information Product #
Description
Pkg. Size
U.S. Price
29920 Disposable Polystyrene ColumnsIdealforpacking0.5–2.0mLbedvolumes.
100/pkg $144
29922 Disposable Polypropylene Columns Idealforpacking1–5mLbedvolumes.
100/pkg $174
29924 Disposable Polypropylene ColumnsIdealforpacking2–10mLbedvolumes.
100/pkg $190
29923 Disposable Polypropylene Funnels BufferreservoirsthatfitProduct#’s29920,29922and29924.
50/pkg $109
29925 Disposable Column Trial Pack IncludesaccessoriesplustwoeachofProduct#’s29920,29922and29924andoneofProduct#29923.
TrialPack $ 63
Centrifuge Columns
Efficientlyhandleawidevarietyofresinvolumesforaffinitypurification!ThermoScientificPierceCentrifugeColumnsareconvenienttoolsforhandling40μL–10mLofanaffinitypurificationsupport.Addtheaffinityresintooneofthepolypropylenecolumns,removethetwist-offbottomandallowtheresintopackitself.Thenaddyoursampleandallowittobindtothesupport.Useacentrifugetoefficientlywashawayanycontaminantsandeluteyourpurifiedprotein.
PierceCentrifugeColumnsallowyoutouseaspin-columnformatinadditiontotraditionalgravityflowtoreducethetimerequiredforcolumnwashingandelution.Thisacceleratessampleprocessingtimeandmakesmultiple-sampleprocessingpossible.Centrifugecolumnsallowyoutoaffinity-purifymoreproteininlesstime!
Centrifugecolumnsaremadefromlowprotein-bindingpolypropyleneforcompatibilitywithproteinpurification,andtheyfitintostandardcentrifugetubesforuseinanycentrifuge.
Applications for Centrifuge Columns:•Affinitypurification/affinitychromatography•Immunodepletion•Spindesalting
0.8mL Centrifuge ColumnsHighlights:•TotalVolume:800μL•ResinVolume:40–400μL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandardmicrocentrifugetubes (e.g.,Product#69720)•CapType:O-ringscrew-topcap•Twist-offbottom
2mL Centrifuge ColumnsHighlights:•TotalVolume:5mL(2mLresinbed,3mLreservoir)•ResinVolume:2mL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandard15mLconical centrifugetubes•CapType:Screw-topcap•Twist-offbottomandpress-oncaptoreseal
5mL Centrifuge ColumnsHighlights:•TotalVolume:8mL(5mLresinbed,3mLreservoir)•ResinVolume:5mL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandard15mLconical centrifugetubes•CapType:Screw-topcap•Twist-offbottomandpress-oncaptoreseal
10mL Centrifuge Columns Highlights:•TotalVolume:22mL(10mLresinbed,12mLreservoir)•ResinVolume:10mL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandard50mLconical centrifugetubes•CapType:Screw-topcap•Twist-offbottomandpress-oncaptoreseal
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89868 Centrifuge Columns, 0.8mL capacity Includes:PierceCentrifugeColumns ScrewCaps
Kit50each50each
$ 62
89869 Centrifuge Columns, 0.8mL capacity Includes:PierceCentrifugeColumns ScrewCaps
Kit4x50each4x50each
$192
89896 Centrifuge Columns, 2mL capacity Includes:PierceCentrifugeColumns ScrewCapsandTips
Kit25each25each
$ 39
89897 Centrifuge Columns, 5mL capacity Includes:PierceCentrifugeColumns ScrewCapsandTips
Kit25each25each
$ 44
89898 Centrifuge Columns, 10mL capacity Includes:PierceCentrifugeColumns ScrewCapsandTips
Kit25each25each
$ 49
69707 Column Extender Fits89896,89897and89898.Increasescolumncapacityby35mL
10each $ 28
5ml
4ml
3ml
2ml
1ml
10ml 9ml
8ml 7ml
6ml 5ml
4ml 3ml
2ml 1ml
12 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Fusion Protein Purification
CulturesofE. coliorPicchiaarecommonvehiclesforproteinexpression.Theyarelowcostandlowmaintenanceplatformswhichcanbeeasilyscaleduptodeliverproteinatmilligramtogramyields.Thesemicrooganismsarealsoveryeasytotrans-formwithaDNAvectorcontainingthegeneofinterest.Typically,researchersusecommonexpressionvectorswhichpossesstheproperpromoterelementsforexpressionandinclusionofanaffinitytagsequence.TheaffinitytagsequenceisclonedinframewiththeDNAsequenceofthetargetprotein,andwillflankeithertheN-orC-terminus.Thetwomostcommonaffinitytagsarepolyhistidine(6xHis)andglutathione-S-trasferase(GST).
Polyhistidine Purification
Cobalt-histidine binding.
Thepolyhistidinetagisasequenceoffivetoninehistidineaminoacidsattachedtotheterminusofatargetprotein.Thepolyhistidinetagispurifiedusingimmobilizedmetalaffinitychromatography(IMAC).Forhistidinetagpurification,eithernickelorcobaltisimmobilizedontoasolidchromatographyresin.Whilethetwometalscanbeusedinterchangeably,typicallynickelhasahigherbindingcapacitywhereascobaltbindslessnon-specificproteintodeliverapurerfinalprotein.
IMACresinsworkbychargeinteractionswiththenitrogenatomsonthehistidineaminoacidsidechaintobindandimmobilizethehistidine-taggedproteinfromacelllysate.Theincorporationofmultiplehistidineresiduesasanaffinitytagisdesignedtoimprovethischargeassociation.BecauseIMACaffinityforhistidineresi-duesisnotdependentonthesecondarystructureoftheprotein,IMACpurificationcanbeperformedunderdenaturingconditions.IMACpurificationis,however,sensitivetopHandthepresenceofchelatorsandreducingagents.SeeTable1foralistofcommoninterferingagentsandtheirconcentrationtoleranceforbothnickelandcobaltresin.
Fusion Protein Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 13
Nickel-histidine binding.
Table 1. Common interfering agents for Ni-NTA and cobalt resins.
Reagent Tolerance for Ni-NTA resin Tolerance for Cobalt resin
ß-Mercaptoethanol 10mM 10mM
CHAPS 1% 1%
Ethanol 30% 30%
Ethyleneglycol 30% 30%
HEPES 50mM 50mM
Glycerol 20% 20%
Guanidinium 6M 6M
Imididazole 500mM 200mMatpH7.0-8.0forelution
KCl 500mM 500mM
MES 20mM 20mM
MOPS 50mM 50mM
NaCl 1.0M 1.0M
NP-40 1% 1%
SDS 1% 1%
TRIS 50mM 50mM
Triton®-X-100 <1% <1%
Urea 8M 8M
Onceimmobilized,imidazoleisusedtodisruptthechargeattractionsbetweentheimmobilizedmetalaffinitychromatographyresinandthehistidine-taggedprotein.Theelutedhistidine-taggedproteincanbeeasilycleaned-upusingadesaltingcolumnordialysiscassettetoremoveimidazole.Oftentraceamountsofimidazoleareincludedintheproteinbindingandwashstepstocompeteawaybindingofendogenousproteinswithmultiplehistidines.
Glutathione-S-transferase Purification
GST binding.
GSTisa26kDaendogenousenzymefoundinbothprokaryotesandeukaryotes.InthecellGSTdetoxifiesreactiveoxygenspeciessuchasfreeradicalsandperoxides.GSTperformsthistaskbyneutralizingreactiveoxygenspecieswiththeantioxidantglutathione(GSH).GSHisanendogenoustripeptide(Glu-Cys-Gly)containingacysteineresidue,whosesulfhydrylsidechainfunctionsasareducingagent.
GSTmakesanidealaffinitytagbecauseofitsstrongbindingtoreducedglutathione.GSTpossessnumeroustyrosineresiduesintheintheGSHbindingpocket,andoneofthesetyrosineshydrogenbondstothesubstrateglutathioneformingastablecomplex.In vivo,GSTwouldtransferglutathionetoareactiveoxygenspeciesandneutralizeit.
Asaresearchtool,scientistshavetakenadvantageofthestronginteractionbetweenGSTandGSHtoselectivelyextractrecom-binantproteins.Inthisstrategy,glutathioneisimmobilizedontoasolidsupport(typicallyanagarosebead)andtheaminoacidsequencefortheenzymeGSTisclonedintoeithertheC-orN-terminusofthetargetgene(commonlyusingthepGEX®-seriesofexpressionvectors).AfterbindingoftheGST-taggedfusionproteintotheimmobilizedglutathioneagarose,excessreducedglutathioneisintroduced(typicallybetween10-50mM)tocompeteoffthetargetprotein.Again,simpledialysisordesaltingcolumnscanbeusedtocleanupthefinalGST-taggedprotein.Forreasonsthathavenotbeenfullycharacterizedintheliterature,thestruc-tureoftheGSTfusiontagoftendegradesupondenaturationandreductionforproteingelelectrophoresis(e.g.,SDS-PAGE).Asaresult,electrophoresedsamplesoftenappearasaladderoflowerMWbandsbelowthefull-sizedfusionprotein.
Incontrasttopolyhistidinepurification,GSTpurificationrequiresthetargetproteinmaintainnativetertiarystructure.Additionally,theGSTtagisquitelarge(26kDa)comparedtothesixhistidineswhichcompriseatypicalpolyhistidinetag.Tocircumventtheproblemsassociatedwitha26kDaappendage,selectiveproteasesareusedtocleavetheGSTtagfromtheGST-fusionprotein.TheseproteasesincludeFactorXa(Product#32520)orthrombin.
14 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
His-Tagged Protein Purification Resin
A cost-effective Ni-NTA resin is now available.
Theexpressionandpurificationofrecombinantproteinsiscentraltoproteinregulation,structureandfunctionstud-ies.Themajorityofrecombinantproteinsareexpressedasfusionswithshortaffinitytagswiththemostpopularbeingthepolyhistidine(6xHis)tag.ThemethodusedtopurifyrecombinantHis-taggedproteinsisimmobilizedmetalaffinitychromatography(IMAC),consistingofchelatingresinschargedwitheithernickelorcobaltionsthatcoordinatewiththehistidinesidechains.ThenewThermoScientificHisPurNi-NTAResin
complementstheHisPur™CobaltResin.Ni-NTAresinsarethemostcommonIMACresinchoicefor6xHis-taggedproteinpurificationsbecauseofthefourmetal-bindingsitesonthechelate,whichenableshigh-proteinbindingandlow-metalionleaching.
Highlights:• High capacity–bindupto60mgof6xHis-taggedproteinper
milliliterofresin• Versatile–purifyproteinsusingnativeordenaturingconditions• Compatible–usewithThermoScientificCellLysisReagents
andavarietyofbufferadditives• Flexible–availableinmultipleformats,includingbulkresin,spin
columnsandchromatographycartridges•Cost-effective–reusethesamebatchofresinatleastfivetimes•Easy to use–pre-formulatedbuffersavailableforkitformats
His-taggedgreenfluorescentprotein(GFP)wasrecoveredinsimilarorgreaterpurityandyieldcomparedtoothercommerciallyavailablenickel-chelatedresins(Figure1).HisPurNi-NTAResinisalsocost-effectivebecauseitcanbeusedatleastfivetimeswithoutlossinperformance(Figure2).Theresinisamenabletochromatography-cartridgeformat,resultinginasharpelutionpeakevidentinthechromatogram(Figure3).WhenHisPurNi-NTAResiniscomparedtoHisPurCobaltResin(Product#90090)andNi-IDAresinusingthebatch-bindmethod,theresultsdependonproteinexpressionlevel(Table2andFigure4).Whenpurifyingalow-expressionprotein,suchas6xHis-AIF2,thereisadramaticdifferenceinpurity.HisPurCobaltResinyieldsthepurestproteinfollowedbyHisPurNi-NTAResinwithNi-IDAbeingtheleastpure.Whenpurifying6xHis-GFP,ahighexpresser,therewasminimaldifferencebetweenHisPurNi-NTAandCobaltResins.Similarresultswereobtainedusingspinpurification(datanotshown).
M
10
15
2520
37
50
75
100
150
250250kDa
L HisPur Ni-NTA
Supplier Q
Supplier C
Ni-IDA
6xHis-GFP
Figure 1. Thermo Scientific HisPur Ni-NTA resin is comparable to or performs better than other suppliers’ nickel resins.Bacteriallysate(12mgtotalprotein)containingover-expressed6xHis-greenfluorescentprotein(GFP)wasappliedtoHisPurNi-NTAResin(200μL)andpurifiedbythebatch-bindmethodasdescribed.ThesameamountoftotalproteinwasappliedtoSupplierQ,SupplierCandNi-IDAresinsperthemanu-facturers’instructions.Gellaneswerenormalizedtoequivalentvolume.M =molecular-weightmarkerandL =lysateload.
Fusion Protein Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 15
M
First Use
L FT W E
Fifth Use
L FT W E
10
15
25
20
37
50
75
100
150kDa
6xHis-Protein L
Figure 2. Thermo Scientific HisPur Ni-NTA Resin can be used at least five times without losing performance. Bacteriallysate(20mgtotalprotein)con-tainingover-expressed6xHis-ProteinLwasappliedtoHisPurNi-NTAResin(200μL)andpurifiedbythebatch-bindmethodasdescribed.Beforeeachreuse,theresinwaswashedwith20mmMESbuffer,0.1MNaCl;pH5(1mL)andfollowedbyawaterwash(1mL).Gelslaneswerenormalizedtoequivalentvolume.M =molecular-weightmarker,L =lysateload,FT =flow-throughandE =elution.
280 nm485 nm
0
500
1000
1500
2000
2500
3000
3500
mAU
Flow-through
Wash
0 20 40 60 80 100 120mL
6xHis-GFP
Figure 3. Thermo Scientific HisPur Ni-NTA Chromatography Cartridges provide an automatable solution for obtaining high-purity proteins. Bacteriallysate(140mgtotalprotein)containingover-expressed6xHis-GFPwasdilutedwithequilibrationbufferandappliedtoaHisPurNi-NTAChromatographyCartridgeataflowrateof1mL/min.ThecartridgewaswashedwithPBS,60mmimidazoleuntilthebaselineabsorbancewasreached.6xHis-GFPwaselutedwithPBS,300mmimidazole.Elutionwasmonitoredat280nm(blueline)and485nm(redline).
Table 2. Elution fractions were analyzed for protein content using the Thermo Scientific Coomassie Plus (Bradford) Protein Assay Kit (Product # 23236). Purity was determined by analyzing the stained SDS-PAGE gel (Figure 4) with densitometry software.
Sample Resin Total yield (mg) Purity
6xHis-AIF2 HisPurNi-NTA 0.5 32%
Ni-IDA 0.5 25%
HisPurCobalt 0.4 49%
6xHis-GFP HisPurNi-NTA 0.8 90%
Ni-IDA 0.6 52%
HisPurCobalt 0.7 91%
M
10
15
2520
37
50
75
100
150
250kDa
L HisPur N
i-NTA
Ni-IDA
HisPur C
obalt
L HisPur N
i-NTA
Ni-IDA
HisPur C
obalt
6xHis-GFP
6xHis-AIF2
Figure 4. Thermo Scientific HisPur Cobalt Resin produces the most pure results. Bacteriallysatecontainingover-expressed6xHis-AIF2(6mgtotalprotein)or6xHis-GFP(4mgtotalprotein)wasappliedtoHisPurNi-NTAResin(200μL)andpurifiedbythebatch-bindmethodasdescribed.ThesameamountoftotalproteinwasappliedtoNi-IDAandHisPurCobaltResinsandpurifiedasdescribedintheinstructions.Gelslaneswerenormalizedtoequivalentvolume.M =molecular-weightmarker,L =lysateload.
TheHisPurNi-NTAResinoffersacost-effectivealternativetoothercommerciallyavailablenickel-IMACresins,withoutcompro-misingyield,purityorperformance.TheHisPurNi-NTAandCobaltResinsareavailableinmultipleformatstoaccommodateavarietyofapplicationsandpurificationvolumes.
Ordering Information
Product # Description Pkg. SizeU.S. Price
88221 HisPur Ni-NTA Resin 10mL $ 85
88222 HisPur Ni-NTA Resin 100mL $ 579
88223 HisPur Ni-NTA Resin 500mL $2275
88224 HisPur Ni-NTA Spin Columns, 0.2mL 25columns $ 155
88225 HisPur Ni-NTA Spin Columns, 1mL 5columns $ 99
88226 HisPur Ni-NTA Spin Columns, 3mL 5columns $ 150
88227 HisPur Ni-NTA Purification Kit§, 0.2mL 25columns $ 269
88228 HisPur Ni-NTA Purification Kit§, 1mL 5columns $ 253
88229 HisPur Ni-NTA Purification Kit§, 3mL 5columns $ 269
90098 HisPur Ni-NTA Chromatography Cartridges, 1mL
5cartridges $ 155
90099 HisPur Ni-NTA Chromatography Cartridges, 5mL
2cartridges $ 200
§ For complete ordering information and kit components, please visit www.thermoscientific.com/pierce
16 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Cobalt Resin, Spin Columns and Chromatography Cartridges
Specific, fast and gentle purification of His-tagged proteins.
ThepreferredmethodforpurifyingrecombinantHis-taggedproteinsisimmobilizedmetalaffinitychromatography(IMAC).Traditionally,chelatingchromatographyresinsarechargedwitheithernickelorcobaltionsthatcoordinatewiththehistidinesidechainsinthe6xHis-tag.HisPurCobaltResinisatetradentatechelatingresinchargedwithcobaltthatbindsHis-taggedproteinswithhighspecificityandreleasesthemunderlowerimidazoleconcentrationsthanrequiredwithnickelresins(seeFigure5).HisPurCobaltResincanbeusedtoobtainhigh-purityproteinswithnometalcontamination.
TheHisPurChromatographyCartridgesareconvenient,reliableandready-to-usepre-packeddevicesfortheisolationofproteinsinsolutionandpurificationofHis-taggedfusionproteins.TheHisPurCartridgesarecompatiblewithautomatedLCinstrumen-tation,suchastheÄKTA®andBioLogicSystems,andadapttomanualsyringeprocessing.
Wash Elution
16.5
2532
47
110
210kDa M F 1 2 3 1 2 3 4 5 L
6xHis-GFP
Thermo Scientific HisPur Cobalt Resin is specific for His-tagged proteins and allows mild, efficient elutions.Bacteriallysate(1.0mgtotalprotein)wasappliedtoa200μLbedvolumeofHisPurCobaltResininaspincolumn.Gellaneswerenormalizedtoequivalentvolume.M =MolecularWeightMarkers(Product#26691),L =lysateloadandF =flow-through.
Highlights:•High purity–obtainpureproteinwithoutoptimizingimidazole washingconditions•Specificity–cobalt:chelatebindingcorebindsfewercontaminants, resultinginlowerbackgroundthannickel(seeTable3)•Low metal leaching–nometalcontaminationinelutedsample•Versatility–purifyproteinsundernativeordenaturingconditions; compatiblewithcelllysisreagentsandavarietyofbufferadditives•Flexibility–availableasbulkresinorpredispensedcolumns compatiblewithbothspinandgravity-flowformats•Cost effective–reuseordiscard•Superior–performsbetterthanothercommerciallyavailable IMACResins(seeFigure5)
Co2+
1
GFP β-gal Protein L
2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6 L
Ni2+ Ni2+Co2+ Ni2+Co2+
Figure 5. Thermo Scientific HisPur Cobalt Resin outperforms other IMAC resins. ComparableyieldandhigherpurityobtainedwithHisPurCobaltResin(lane1)comparedtootherIMACresins(lanes2to6).Celllysatescontainingover-expressedrecombinant6xHis-taggedproteinwerepreparedinB-PERBacterialProteinExtractionReagent(Product#78243)andProteaseInhibitors(Product#78410).ProteinconcentrationsweredeterminedbyCoomassiePlusProteinAssay(Product#23238).E. coli lysatescontainingover-expressedHis-taggedGFP, β-galactosidaseorProteinLwereappliedto0.2mLbedvolumesofeachIMACresininspincolumnformat.Binding,washandelutionbufferswerepreparedandusedpereachmanufacturers’instructions.ThefirstelutionfractionforeachIMACresinwasanalyzedbySDS-PAGEandproteinpuritydeterminedbydensitometry.Gellaneswerenormalizedtoequivalentvolume.Lanes: 1=HisPurCobaltResin,2=supplierCcobaltresin,3=supplierScobaltresin,4=supplierGnickelresin,5=supplierQnickelresin,6=Ni-IDAandL=lysateload.
Fusion Protein Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 17
Table 3. Thermo Scientific HisPur Cobalt Resin yields more His-tagged protein and higher purity than other Co2+ and Ni2+ IMAC Resins.
His-GFP His-β-Gal His-Protein L
Yield(μg)* Purity(%)** Yield(μg)* Purity(%)** Yield(μg)* Purity(%)**
ThermoScientificHisPurCobaltResin 298 87 78 93 42 77
SupplierCCobaltResin 206 78 26 90 35 76
SupplierSCobaltResin 211 85 27 65 38 77
SupplierGNickelResin 239 84 42 83 29 68
SupplierQNickelResin 242 85 24 48 30 72
Ni2+-IDAResin 70 37 6 16 17 46
* Recovered from a 5mg total protein load (total protein yield x purity). ** Purity of the first elution fraction.
HisPur Cobalt Cartridge Performance Data:
0
1000
2000
3000
4000
5000
6000
0 5 10 15 20 25Chromatography Progress (mL)
Flow-through Wash Elution
Abs
orba
nce
280n
m (m
AU
)
Flow-through (FT)M S Wash Elution
Electrophoresed Fractions
Chromatography Profile
Purification of 6xHis-GFP from E. coli lysate using a Thermo Scientific HisPur Cobalt Cartridge. His-taggedgreenfluorescentprotein(GFP)wasextractedfromE. coliusingThermoScientificB-PERBacterialProteinExtractionReagentinPhosphateBuffer(Product#78266)containingHaltProteaseInhibitorCocktail,EDTA-Free(Product#78415).Thelysatewasdiluted1:1withequilibration/washbuffer(50mMsodiumphosphate,300mMsodiumchloride,10mMimidazole,pH7.4)andappliedtoaThermoScientificHisPurCobaltChromatographyCartridgeataflowrateof0.3mL/min.Thecartridgewaswashedwithequilibration/washbufferuntilthebaselineabsorbanceatA280wasreached.His-taggedGFPwaseluted(50mMsodiumphosphate,300mMsodiumchloride,150mMimidazole;pH7.4)andselectedfractionswereanalyzedbySDS-PAGEandThermoScientificGelCodeBlueStainReagent(Product#24592).M =MWMarker;S=non-fractionatedlysate;FT =flow-through.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89967 HisPur Cobalt Spin Columns, 0.2mL 25columns $ 160
89968 HisPur Cobalt Spin Columns, 1mL 5columns $ 105
89969 HisPur Cobalt Spin Columns, 3mL 5columns $ 155
89964 HisPur Cobalt Resin 10mLbottle $ 88
89965 HisPur Cobalt Resin 100mLbottle $ 600
89966 HisPur Cobalt Resin 500mLbottle $2347
90090 HisPur Purification Kit, 0.2mL 25columns $ 277
90091 HisPur Purification Kit, 1mL 5columns $ 262
90092 HisPur Purification Kit, 3mL 5columns $ 277
90093 HisPur Chromatography Cartridges 5x1mL $ 160
90094 HisPur Chromatography Cartridges 2x5mL $ 267
18 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Fusion Protein Purification
High-quality purification of GST-fusion proteins
Multiple formats available to meet your purification needs
PurificationofglutathioneS-transferase(GST)fusionproteinsusingglutathioneagarosebeadsprovidesone-step,high-purityaffinitypurification.TheThermoScientificPierceGlutathioneAgaroseiscomposedof6%crosslinkedbeadedagarosewithglutathione(GSH)immobilizedbyitscentralsulfhydryl.GST-fusionproteinsarepurifiedwithhighyieldbecauseofthe12-atomGSHlinkerthatminimizessterichindrance.ThePierceGlutathioneAgaroseisavailableinresinslurrypackages,spincolumns,completepurificationkitsandFPLC-readychromatographycartridges.
Highlights:• High capacity–bindsatleast40mgofpureGSTpermilliliter
ofresin• Cost-effective–resiniseconomicallypricedandcanbereused
atleastfivetimeswithoutreductioninperformance• High yield and purity–eachmilliliterofresincanpurify≥ 10mg
ofGST-taggedproteinfromlysateswith>90%purity• Compatible–validatedandeffectivewithThermoScientific
CellLysisReagents• Versatile–purifylysatesorpulldownprotein-proteininteractions• Flexible–availableinmultipleformats
GlutathioneS-transferaseandpolyhistidinearethemostcommonlyusedproteintagsforaffinitypurification.GSTproducescleanerpurificationsthanhistidinetagsandstabilizesoverexpressedfusionproteins.GSTbindsspecificallytoreducedGSHinnear-neutral,nondenaturingconditions.TheboundGST-taggedproteiniseasilydissociated(eluted)fromGSHresinbycompetitivedisplacementwithabuffercontainingfree,reducedGSH.Alternatively,ifthefusionproteinisdesignedwithaproteasecleavagesite,itmaybereleased(cleaved)fromthecolumnattheGSTtagjunctionusingthrombin,HRV3CproteaseorFactorXa.
ThePierceGlutathioneAgaroseishighlyeffectiveforpurifyingavarietyofGST-fusionproteins(Figure6).ThepurityandyieldofGST-fusionproteinrecoveryiscomparabletoorbetterthanglutathioneresinsfromothersuppliers(Figure7).Theresiniscomposedof6%crosslinkedbeadedagarose,makingitastructurallystableandresilientaffinitysupportthatenablesrepeateduses.ThePierceGlutathioneAgaroseisalsoamenabletochromatographycartridgeformat,producingbaselineresolutionandasharpelutionpeak(Figure8)withnovisibleresinbedcompression.
GST-Pak1
kDa M L FT W1 W2 W3 E1 E2 E3
210
11080
47
3225
16.5
GST-ERK
M L FT W1 W2 W3 E1 E2 E3kDa
210
11080
47
3225
16.5
GST-Bid
M L FT W1 W2 W3 E1 E2 E3kDa
210
11080
47
3225
16.5
Figure 6. The Thermo Scientific Pierce Glutathione Agarose effectively purifies different GST-fusion proteins.ThreeGST-fusionproteinswereexpressedinE. coliandextractedwithThermoScientificB-PERReagentwithEnzymes(Product#90078).GST-Pak1andGST-Bidwerepurifiedusingthespinformat,andGST-ERKwaspurifiedbygravityflow.GST-Pak1andGST-Bid,2.4mgand11mgoftotalprotein,respectively,werepurifiedwitha0.2mLPierceGlutathioneSpinColumn(Product#16106)perkitinstructions.ForGST-ERK,11.2mgoftotallysateproteininbuffer(50mMTris,150mMNaCl;pH8.0)wasappliedtoa0.5mLbedofPierceGlutathioneAgarose(Product#16100)inadripcolumnandelutedwith125mMTris,150mMNaCl,10mMglutathioneatpH8.0.Chromatographyfractionsweresep-aratedbySDS-PAGEandstainedwithThermoScientificGelCodeBlueStainReagent(Product#24590).M =Molecularweightmarkers;L =Lysateload; FT =Flow-through;W =Wash;E =Elution.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 19
kDa
250150100
755027
25201510
M L FT
ThermoScientific
GEHealthcare Qiagen Clontech Sigma
E FT E FT E FT E FT E
VendorThermo
ScientificGE
Healthcare Qiagen Clontech Sigma
Yield 537μg 562μg 285μg 299μg 410μg
Purity 93% 93% 90% 91% 94%
Figure 7. Thermo Scientific Pierce Glutathione Agarose delivers high yield and high-purity GST-fusion proteins.E. coli lysate(14.4mgtotalprotein)con-taininganoverexpressedGST-fusionproteinwasincubatedwith50μLGSHresinfromvarioussuppliersandpurifiedpermanufacturers’instructions.TheamountofGSTelutedfromtheresin(yield)wasquantifiedbyThermoScientificCoomassiePlusProteinAssay.Puritywasassessedbydensitometryofthestainedgellanes.M=Molecularweightmarkers;L=Lysateload;FT=Flow-through;E=Elution.
0
0.0 10.0 20.0 30.0 40.0 50.0 ml
mAU
4000
Flow-through
Wash Elution
3000
2000
1000
Figure 8. The glutathione chromatography cartridges provide an automatable solution for GST-fusion protein purification.ClarifiedE. colilysate(50mg)containingoverexpressedGST-Pak1(34kDa)wasappliedtoa1mLglutathionechromatographycartridgeinbuffer(50mMTris,150mMsodiumchloride;pH8.0)ataflowrateof0.5mL/minuteusinganÄKTApurifier®System.Thecartridgewaswashedwiththesamebufferat1mL/minute.GST-Pak1waseluted(50mMTris,150mMsodiumchloride,10mMglutathione;pH8.0)at1mL/minute.
Ordering InformationSee catalog or www.thermoscientific.com/pierce for a complete description of products and kits.
Product # Description Pkg. SizeU.S. Price
16100 Pierce Glutathione Agarose 10mL $ 180
16101 Pierce Glutathione Agarose 100mL $ 975
16102 Pierce Glutathione Agarose 500mL $3000
16103 Pierce Glutathione Spin Columns, 0.2mL 25columns $ 175
16104 Pierce Glutathione Spin Columns, 1mL 5columns $ 175
16105 Pierce Glutathione Spin Columns, 3mL 5columns $ 315
16106 Pierce GST Spin Purification Kit, 0.2mL 25-columnkit $ 275
16107 Pierce GST Spin Purification Kit, 1mL 5-columnkit $ 275
16108 Pierce GST Spin Purification Kit, 3mL 5-columnkit $ 375
16109 Pierce Glutathione Chromatography Cartridges, 1mL
5cartridges $ 230
16110 Pierce Glutathione Chromatography Cartridges, 5mL
2cartridges $ 400
20 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
GST- and PolyHis-Tagged Pull-Down Assay Kits
Prepare to discover a new protein:protein interaction with your GST- or polyHis-tagged bait protein.
Identifyingandcharacterizingtheinteractionsofagivenproteinhasemergedasthemostvaluableinformationthatcanbedevelopedinthepost-genomicera.ThermoScientificPiercePull-DownKitscontainthenecessarycomponentstocaptureinteractingproteinsusingthepopularpull-downmethod.Theonlyitemyouprovideisanappropriatelytaggedfusionproteinasthe“bait.”OurPull-DownKitsaredesignedtoteachthemethodtothefirst-timeuserandtoshortenthetimetoaresultforthoseexperiencedinthismethod.
Step 3. Bind “prey” protein to immobilized “bait” protein.
Step 4. Wash away unbound protein. Step 5. Elute protein:protein interaction complex.
Step 2. Wash away unbound protein.Step 1. Immobilize the fusion-tagged “bait” from the lysate.
Step 6. Analyze protein:protein interaction complex on SDS-PAGE.
= Fusion Tag (GST or polyHis)
Bait
Prey
Marker PurifiedProtein:Protein
Interaction
AgaroseResin
Control
PurifiedBait
Displaced Interacting Complex
P re y P ro te inBait Protein Prey Protein
Bait Protein
Prey Protein-Containing Lysate
Bait Protein
Bait Protein
Bait Protein-Containing Lysate
Affinity Ligand(Glutathione or Co2+ Chelate)
AgaroseBead
Prey Protein
Bait Protein Prey Protein
Bait Protein Prey Protein
Glutathioneor
Imidazole
Spin
Spin
Highlights:• Providesacomplete,affordableandeasy-to-usestrategy
fordiscoveryofprotein:proteininteractions• Usescommonlaboratoryequipment(e.g.,microcentrifuges
andmini-gels)•Adaptstosingle-ormultiple-sampledemands•Suppliedcompletewithcelllysisbuffer• Flexibleprotocolaidsinthecaptureofweakortransient
interactions• Efficientrecoveryofinteractingcomplexes
Applications:•Discoveranewprotein:proteininteractionfromacelllysate• Confirmaputativeinteractionfromacelllysateorwitha
previouslypurifiedprotein• Extractprotein:proteininteractioninformationfrom in vitro
transcription/translationlysates
Generalized scheme for use of a Thermo Scientific Pierce Pull-Down Protein:Protein Interaction Kit using a GST-tagged or PolyHis-tagged protein as the “bait.”
Fusion Protein Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 21
Ordering Information Product #
Description
Pkg. Size
U.S. Price
21516 Pierce Pull-Down GST Protein Interaction Kit Sufficient materials for performing 25 pull-down assays using a GST-tagged protein as the bait.Includes:ImmobilizedGlutathione LysisBuffer Glutathione BupHTrisBufferedSaline SpinColumnsAccessoryPack CollectionTubesandCaps AccessoryPack
Kit
750μLsettledgel250mL1g1pack(500μL)27columns100tubes
$320
AcknowledgmentWegratefullyacknowledgeDr.YigongShiofPrincetonUniversityforsupplyingtheGST-BIR2-and9xHisSmac/DIABlO-expressingclones.Dr.Shi’slaboratoryisengagedinresearchaimedatunderstandingthestructuralandmolecularmechanismsinvolvedintumorigenesisandapoptosis.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
21277 Pierce Pull-Down PolyHis Protein Interaction Kit Sufficient materials for performing 25 pull-down assays using a polyhistidine-tagged protein as the bait.Includes:ImmobilizedGlutathione LysisBuffer 4MImidazoleStockSolution BupHTrisBufferedSaline SpinColumnsAccessoryPack CollectionTubesandCaps AccessoryPack
Kit
750μLsettledgel250mL5mL1pack(500μL)27columns100tubes
$320
References1.Chai,J.,et al.(2000).Nature406,855-862.2.Kaelin,W.G.,et al.(1991).Cell64,521-532.3.Sambrook,J.andRussell,D.W.(2001).Molecular Cloning: A Laboratory Manual
3rd Edition.Chapter18:ProteinInteractionTechnologies,Protocol#3:DetectionofProtein-ProteinInteractionsusingtheGSTFusionProteinPull-DownTechnique.ColdSpringHarborLaboratoryPress.
4.Soutoglou,E.,et al. (2000).Mol. Cell 5, 745-751.
Lane # A. GST-Tag Pull-Down B. PolyHis-Tag Pull-Down
1 LysatefromE. coliexpressingGST-taggedBIR2(bait protein). LysatefromE. coliexpressing9xHis-taggedwild-typeSmac(bait protein).
2 Flow-throughfromthelysateinLane1boundtoanimmobilizedreducedglutathionesupportfor1hourat4°C.
Flow-throughfromthelysateinLane1boundtoanimmobilizedcobaltchelatesupportfor1hourat4°C.
3 Wash#1ofthesupport. Wash#1ofthesupport.
4 Wash#2ofthesupport.(Washes3-5notshown.) Wash#2ofthesupport.(Washes3-5notshown.)
5 LysatefromE. coli expressing9xHis-taggedwild-typeSmac(prey protein).
LysatefromE. coliexpressingGST-taggedBIR2(prey protein).
6 Flow-throughfromthelysateinLane5isallowedtointeractwiththeimmobilizedbaitfor1hourat4°C.
Flow-throughfromthelysateinLane5isallowedtointeractwiththeimmobilizedbaitfor1hourat4°C.
7 Wash#1ofthesupport. Wash#1ofthesupport.
8 Wash#2ofthesupport.(Washes3-5notshown.) Wash#2ofthesupport.(Washes3-5notshown.)
9 Baitcontrol.BaittreatedasdescribedinLanes1-8andsubsequentlyeluted.Nopreyadded–justbindingbuffer.
Baitcontrol.BaittreatedasdescribedinLanes1-8andsubsequentlyeluted.Nopreyadded–justbindingbuffer.
10 Preycontrol.PreytreatedasdescribedinLanes1-8andsubsequentlyeluted.Nobaitadded–justbindingbuffer.
Preycontrol.PreytreatedasdescribedinLanes1-8andsubsequentlyeluted.Nobaitadded–justbindingbuffer.
11 Elutionofbait:preycomplex(preparedinLanes1-8)fromthesupportwith100mmreducedglutathione.WesternblottingconfirmsthattheminorbandsobservedinLanes9and11aredegradationproductsofGST-taggedBIR2.
Elutionofbait:preycomplex(preparedinLanes1-8)fromthesupportwith250mmimidazole.
Validation of the Thermo Scientific Pierce Pull-Down Protein Interaction Kits using a known interacting pair.
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11A. B.
22 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Covalent Immobilization of Ligands
Affinity chromatography uses the specific interactions between two molecules for the purification of a target molecule. In practice, a ligand having affinity for a target molecule is covalently attached to an insoluble support and functions as bait for capturing the target from complex solutions.
Theaffinityligandcanbevirtuallyanymoleculethatcanspecificallybindthetargetwithoutdisplayingsignificantnonspecificbindingtowardothermoleculesinthesolution.Ligandsthathavebeenusedforaffinityseparationsincludesmallorganiccompoundsthatareabletodockintobindingsitesonproteins,inorganicmetalsthatformcoordinationcomplexeswithcertainaminoacidsinproteins,hydrophobicmoleculesthatcanbindnonpolarpocketsinbiomole-cules,proteinswithspecificbindingregionsthatareabletointeractwithotherproteins,andantibodies,whichcanbedesignedtotargetanybiomoleculethroughtheirantigen-bindingsites.
Theconceptofusingimmobilizedaffinityligandstotargetbiomoleculeshasextendedbeyondchromatographicapplications.Affinityligandsarenowcoupledtolatexbeads,nanoparticles,macro-beads,membranes,microplates,arraysurfaces,dipsticksandahostofotherdevicesthatfacilitatethecaptureofspecificbiomolecules.Theapplicationofaffinitytargetingincludespurification,scavenging(orremovalofcontaminants),catalysis(ormodificationoftargetmolecules)andabroadrangeofanalyticalusestoquantifyatargetmoleculeinasamplesolution.
Designingcustomaffinitysupportsthatareabletotargetuniquebiomoleculesrequiresmethodstocovalentlylinkaligandtoaninsolublematrix.Regardlessoftheintendedapplication,thechemicalreactionsthatmakeligandattachmentpossiblearewellcharacterizedandfacilitatetheattachmentofbiomoleculesthroughtheircommonchemicalgroups.Thetypesoffunctionalitiesgenerallyusedforattachmentincludeeasilyreactivecomponentssuchasprimaryamines,sulfhydryls,aldehydes,carboxylicacids,hydroxyls,phenolicgroupsandhistidinylresidues.Usually,thesolid-phasematrixfirstisactivatedwithacompoundthatisreactivetowardoneormoreofthesefunctionalgroups.Theactivatedcomplexthencanformacovalentlinkagebetweentheligandandthesupport,resultinginligandimmobilization.
Thetypeoflinkagethatisformedbetweenthematrixandtheimmobilizedligandaffectstheperformanceoftheaffinitysupportinanumberofways.Alinkagethatallowsthecoupledligandtoleachfromthematrixwillresultincontaminationofthepurifiedproteinandshortentheusefullifeoftheaffinitysupport.Alinkagethatintroducesachargedfunctionalgroupintothesupportcancausenonspecificbindingbypromotingion-exchangeeffects.Alinkagethataltersthestructureofthematrixcanchangetheflowandbindingcharacteristicsofthesupport.Cyanogenbromide(CNBr)-activatedsupportsareinformativeasanexampleoftheseprinciples.Thispopularimmobilizationmethodresultsinalinkagethat:
1.Hasaconstantleakageofligandfromthematrixthatbecomesacontaminantinthepurifiedpreparation.
2.Includesachargedisoureagroupinthelinkage,resultinginnonspecificbinding.
3.Causesextensivecrosslinkingofthematrix,reducingtheabilityoflargemoleculestopenetrateintotheinterioroftheresin.
Weofferanumberofactivatedaffinitysupportsthataredesignedtocoupleligandsofeverytypeviastable,unchargedcovalentlinkagesthatavoidintroducingundesirablepropertiesintothesupports.Theactivationchemistryandprotocolshavebeenoptimizedtoensureexcellentcouplingyieldswithminimaleffortunderavarietyofconditions.Eachactivatedsupportcomeswithinstructionsforuseandliteraturereferencesasexamples.Theassociatedkitscontainallthecouplingbuffers,washbuffersandcolumnsnecessarytoperformtheligandimmobilizationandproduceasupportreadytoperformanaffinityseparation.
Covalent Coupling of Affinity Ligands to Chromatography Supports
22
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 23
Coupling Affinity Ligands through Amine Groups
Themostcommonfunctionaltargetforimmobilizingproteinmoleculesistheaminegroup,whichispresentonthevastmajorityofproteinsbecauseoftheabundanceoflysinesidechaine-aminesandN-terminala-amines.ThermoScientificAminoLinkCouplingResinandAminoLinkPlusCouplingResinarepreparedfromcrosslinkedagarosesupports,andtheyaredesignedtocreateastablelinkagebetweenaminegroupsandthesupportmaterial.AminoLink®Resinsareactivatedtocontainnumerousaldehydegroups,whichcanbeusedtoimmobilizeamine-containingligandsbyreductiveamination.
TheimmobilizationreactionusingreductiveaminationinvolvestheformationofaninitialSchiffbasebetweenthealdehydeandaminegroups,whichthenisreducedtoasecondaryaminebytheadditionofsodiumcyanoborohydride.Thecyanoborohydridereducingagentusedduringthecouplingprocessismildenoughnottocleavedisulfidesinmostproteins,anditwillnotreducethealdehydereactants–onlytheSchiffbaseintermediates.Itisbesttoavoidstrongerreducingagentssuchassodiumborohydridebecauseofthepotentialfordisulfidereductionoftheproteinandreductionofthealdehydesonthesupporttohydroxyls,effectivelyquenchingthereaction.Dependingonthetypeandamountofligandpresent,acouplingreactionusingreductiveaminationcanachieveimmobilizationyieldsofgreaterthan85%.
H
H2N–R
NaCNBH3
O N H
R
Aldehyde Group onThermo Scientific
AminoLink Coupling ResinAffinity Ligand Coupled
via Secondary Amine Bond
Anotheramine-reactivestrategythatcanbeusedforimmobilizationistheazlactoneringpresentinUltraLinkBiosupport.Aprimaryaminewillreactwithanazlactonegroupinaring-openingprocessthatproducesanamidebondattheendofafive-atomspacer.Thegroupisspontaneouslyreactivewithamines,requiringnoadditivesorcatalyststodrivethecouplingprocess.TheUltraLinkBiosupportissupplieddrytoensurestabilityoftheazlactonegroupsuntiluse.Addingaquantityofthesupporttoasamplecontainingaproteinorotheramine-containingmoleculecausesimmobilizationtooccurwithinaboutonehour.Forproteinimmobilizationathighyield,itisrecommendedthatthecouplingbuffercontainalyotropicsalt,whichfunctionstodrivetheproteinmoleculestowardthebeadsurface.Thisbringsthehydrophilicaminescloseenoughtotheazlactoneringstoreactquickly.ThesimplenatureofcouplingaffinityligandstotheUltraLinkBiosupportalongwithitsinherentlylownonspecificbindingmakesitoneofthebestchoicesforimmobilization.
O
N
O
CH3
CH3 NH
OHN
O
R
Azlactone Group onThermo Scientific
UltraLink BiosupportAffinity Ligand Coupled
via Amide Bond
H2N—R
Thermo Scientific UltraLink Biosupport binding capacity for various proteins.
Capacity Protein Coupling Buffer
35.0mg/mL Myoglobin 0.1MCHES,1.0Msodiumcitrate,pH9.0
21.5mg/mL PenicillinAcylase 0.1Msodiumphosphate,1.1Msodiumsulfate,pH7.4
20.9mg/mL a-chymotrypsin 0.1Mborate,1.5sodiumsulfate,pH9.0
35.5mg/mL BSA 0.1Mborate,1.5sodiumsulfate,pH9.0
29.8mg/mL Lysozyme 0.1Mborate,1.0sodiumsulfate,pH9.0
21.0mg/mL HumanIgG 0.1Mborate,1.5sodiumsulfate,pH9.0
Athirdoptionforimmobilizingamine-containingaffinityligandsistheuseofcarbonyldiimidazole(CDI)toactivatehydroxylsonagarosesupportstoformreactiveimidazolecarbamates.Thisreactivegroupisformedonthesupportinorganicsolventandstoredasasuspensioninacetonetopreventhydrolysis.Reactionofthesupportinanaqueouscouplingbufferwithprimaryamine-containingligandscauseslossoftheimidazolegroupsandformationofcarbamatelinkages.ThecouplingprocessoccursatbasicpH(8.5–10),butitisaslowerreactionwithproteinsthanreductiveaminationorazlactonecoupling.ThermoScientificPierceCDISupportsareavailablewiththeCDI-activatedgroup,andtheyareparticularlyadeptatimmobilizingpeptidesandsmallorganicmolecules.Thereactionalsocanbedoneinorganicsolventtopermitcouplingofwater-insolubleligands.
Reactive Imidazole Carbamate on Thermo Scientific Pierce
CDI SupportsAffinity Ligand Coupled
via Carbamate Bond
O
NH
O
RNO O NH2N—R
NHS-ActivatedAgaroseresinusessafe,reliableNHS-esterchemistryanddoesnotrequirehazardouschemicalsforimmobilization.Otheramine-reactivesupports,suchasperiodate-oxidizedresins,usetoxicsodiumcyanoborohydridetostabilizethereactionlinkagetoprimaryaminesandtake4to6hourstocomplete.Traditionalmethodssuchascyanogenbromide-activatedsupportsalsocoupleamines;however,thischemistryresultsinnonspecificbindingandconstantslowleakageofthecoupledligand.ThePierceNHS-ActivatedAgaroseusesthestableNHS-esterchemistrythatallowsimmobilizationreactionstobecompletedinlessthan1hour.
TheNHS-ActivatedAgarosecouplingreactionisperformedinanamine-freebufferatpH7-9.Proteincouplingefficiencyistypicallygreaterthan80%,regardlessoftheligand’smolecularweightorpI.Oncealigandisimmobilized,thepreparedresincanbeusedformultipleaffinitypurificationprocedures.Thecrosslinkedbeadedagarosehasfastlinearflowpotential,makingitusefulforgravity-flowandlow-tomedium-pressureapplications.
24 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
NHS-ActivatedAgaroseresinisavailableinananhydrousacetoneslurryandasdrypowder.Theuniquedryformdoesnotrequirestorageinorremovalanddisposaloftheacetonesolvent.Inaddition,thedryresinisidealforcouplingreactionswithdilutesamplesbecauseitconcentratesthesampleastheresinswells,reducingthevolumeofthestartingmaterialandresultinginhighlyefficientligandimmobilization.
O NH
O
O
O
O
O
N
NH2 –R
O NH
O
O
R
HN
Coupling Affinity Ligands through Sulfhydryl Groups
Itisoftenadvantageoustoimmobilizeaffinityligandsthroughfunctionalgroupsotherthanjustamines.Inparticular,thethiolgroupcanbeusedtodirectcouplingreactionsawayfromactivecentersorbindingsitesoncertainproteinmolecules.Becauseaminesoccuratmanypositionsonaprotein’ssurface,itisusuallydifficulttopredictwhereanamine-targetedcouplingreactionwilloccur.However,ifsulfhydrylgroupsthattypicallyarepresentinfewernumbersaretargetedforimmobilization,thencouplingcanbedoneatdiscretesitesinaproteinorpeptide.Thiolgroups(sulfhydryls)maybeindigenouswithinaproteinmoleculeortheycanbeaddedthroughthereductionofdisulfidesorthroughtheuseofvariousthiolationreagents.Sulfhydrylsalsocanbeaddedtopeptideaffinityligandsatthetimeofpeptidesynthesisbyaddingacysteineresidueatoneendofthemolecule.Thisensuresthateverypeptidemoleculewillbeorientedonthesupportinthesamewayafterimmobilization.
ThermoScientificSulfoLinkCouplingResinisdesignedtoefficientlyreactwiththiol-containingmoleculesandimmobilizethemthroughathioetherlinkage.Thesupportcontainsaniodo-acetylgroupattheendofalongspacerarm,whichreactswithsulfhydrylsthroughdisplacementoftheiodine.OptimalconditionsforthereactionareanaqueousenvironmentatslightlybasicpH,whereinaminesarenotveryreactivetowardtheiodoacetylfunction,butthiolsarehighlyreactiveduetotheirincreasednucleophilicity.Thethioetherbondthatisformedprovidesastablelinkagetoanysulfhydryl-containingmolecule.
HS R
Reactive Iodoacetyl Groupon Thermo ScientificSulfoLink Supports
Affinity Ligand Coupled via
Thioether Bond
HN
O
SR
HN
O
I
Coupling Affinity Ligands through Carbonyl Groups
Mostbiologicalmoleculesdonotcontaincarbonylketonesoraldehydesintheirnativestate.However,itmightbeusefultocreatesuchgroupsonproteinstoformasiteforimmobilizationthatdirectscovalentcouplingawayfromactivecentersorbindingsites.Glycoconjugates,suchasglycoproteinsorglycolipids,usuallycontainsugarresiduesthathavehydroxylsonadjacentcarbonatoms,whichcanbeperiodate-oxidizedtocreatealdehydes.Controlledoxidationusing1mmsodiummeta-periodateat0°Cwillselectivelyoxidizesialicacidgroupstoformanaldehydefunctionalityoneachsugar.Usinghigherconcentrationsofperiodate(10mm)atroomtemperaturewillresultinoxidationofothersugardiolstocreateadditionalformylgroups.Aldehydesonthecarbohydrateportionofglycoconjugatescanbecovalentlylinkedwithaffinitysupportsthroughanimmobilizedhydrazide,hydrazineoraminegroupbySchiffbaseformationorreductiveamination.
ThermoScientificCarboLinkCouplingResincontainslongspacerarmsthatterminateinhydrazidegroups.Reactionofthehydrazideswithaldehydesformshydrazonelinkages,whichareaformofSchiffbasedisplayingbetterstabilitythanthoseformedbetweenanamineandanaldehyde.TheCarboLink™Resincanbeusedtoimmobilizeglycoproteins,suchasantibodies,afterperiodateoxidationofthecarbohydrate.CouplingantibodiesinthismannerspecificallytargetstheheavychainsintheFcportionofthemolecule.Sincethisisawayfromtheantigen-bindingsitesattheendoftheFvregions,immobilizationusingthisrouteoftenresultsinthebestretentionofantigen-bindingactivity.
NH
O
NH2
NH
O
N R
Reactive Hydrazide Group on Thermo Scientific
CarboLink Supports
Affinity LigandCoupled via
Hydrazone Bond
R H
O
Covalent Coupling of Affinity Ligands to Chromatography Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 25
TheCarboLinkResinalsocanbeusedtocouplecarbohydratesandsugarsthroughtheirreducingends.Aldehyde-orketone-containingsugarswillreactwiththeimmobilizedhydrazidegroupswithoutoxidationofothersugarhydroxyls.However,thisreactionmaybedramaticallyslowerthancouplingwithoxidizedsugarsbecausethesenativealdehydesorketonesareusuallytiedupinacetalorketalringstructures.Theseringscanopeninaqueoussolutiontorevealthealdehydeorketone,buttheopenstructureispresentonlyasmallpercentageofthetime.Thus,thereducingendsofsugarshavedecreasedreactivitytowardanimmobilizedhydrazide,sometimesrequiringdaysofreactiontimetoobtainacceptableimmobilizationyields.
Althoughthehydrazonebondcreatedbetweentheimmobilizedhydrazideandanaldehydeismuchmorestablethanamine-aldehydeSchiffbases,toobtainaleach-resistantlinkageitisrecommendedthattheSchiffbasebereducedwithsodiumcyanoborohydride.Thisisespeciallytrueifaligandiscoupledthathasonlyasinglepointofattachmenttothesupport.Reductionofthehydrazonecreatesastablebondthatwillperformwellinaffinitychromatographyapplications.
Affinity LigandCoupled via
Hydrazone Bond
Stable LigandLinkage
NH
O
N R
NH
OH
RN
NaCNBH3
Coupling Affinity Ligands through Carboxyl Groups
Thecarboxylgroupisafrequentconstituentofmanybiologicalmolecules.Particularly,proteinsandpeptidestypicallycontainnumerouscarboxylicacidsduetothepresenceofglutamicacid,asparticacidandtheC-terminala-carboxylategroup.Carboxylicacidsmaybeusedtoimmobilizebiologicalmoleculesthroughtheuseofacarbodiimide-mediatedreaction.Althoughnoactivatedsupportcontainsareactivegroupthatisspontaneouslyreactivewithcarboxylates,chromatographysupportscontainingamines(orhydrazides)maybeusedtoformamidebondswithcarboxyl-ates.Moleculescontainingcarboxylatesmaybeactivatedtoreactwithanimmobilizedamine(orhydrazide)throughreactionwiththewater-solublecarbodiimideEDC.
EDCreactswithcarboxylatestoformanintermediateesterthatisreactivewithnucleophilessuchasprimaryamines.ThereactiontakesplaceefficientlybetweenaboutpH4.5andpH7.5,anditiscompletewithintwotofourhours,dependingonthetemperature.Theintermediateesterissubjecttohydrolysis;therefore,itisbeneficialiftheamine-containingligandtobeimmobilizedisincludedinthereactionmediumuponadditionofEDC,soitcanreactimmediatelywiththeesterasitforms.
ThermoScientificCarboxyLinkCouplingResinortheUltraLinkDADPAResinmaybeusedtoimmobilizecarboxylate-containingligandsbyEDC.CarboxyLink®ResincontainsanineatomspacerarmandUltraLinkDADPAcontainsa12-atomspacerarmtomini-mizesterichindrance.
R
OH
O
Carboxylate-ContainingAffinity Ligand
+
EDC
Immobilized DADPA on Thermo Scientific CarboxyLink Resin
Immobilized Ligand viaAmide Bond Formation
HN
HN
HN
O
R
HN
HN NH2
26 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Products for Immobilizing Ligands through Primary Amines
Thermo Scientific Pierce NHS Ester-Activated AgarosePierceNHS-ActivatedAgaroseResin,availableasaslurryordrypowder,allowsforthesimpleandefficientimmobilizationofproteinstoabeaded-agarosesupport.TheactivatedagarosecontainsN-hydroxysuccinimide(NHS)esterwithaspacerarmofatleast10atomsinlengththatreactswithprimaryaminesform-ingstableamidelinkages.TheNHS-ActivatedAgarosecouplingreactionisperformedinanamine-freebufferatpH7-9,withtypi-calcouplingefficienciesofmorethan85%.Thepreparedresincanbeusedformultipleaffinitypurificationprocedures.Thecrosslinkedbeadedagarosehasfastlinearflowpotential,makingitusefulforgravity-flowandlow-tomedium-pressureapplications.
O NH
O
O
O
O
O
N
O NH
H
O
ON
Aga
rose
Bea
dA
garo
seB
ead
Protein
+ NH2 Protein
Thermo Scientific NHS Ester-Activated Agarose Support immobilization chemistry.
Target: –NH2
Support:6%agarose
Binding capacity: >25mgprotein/mLresin
Time: 30minutes
Ordering Information Product #
Description
Pkg. Size
U.S. Price
26196 Pierce NHS-Activated Agarose, Dry Swellvolume:6-7.5mL/gofdryresin
1g $ 65
26197 Pierce NHS-Activated Agarose, Dry Swellvolume:6-7.5mL/gofdryresin
5g $200
26198 Pierce NHS-Activated Agarose Spin Columns, 0.2mL 25spincolumnscontaining33mgofNHS-ActivatedAgaroseSwellvolume:6-7.5uL/mgofdryresin
25columns $125
26199 Pierce NHS-Activated Agarose Spin Columns, 2mL 5spincolumnscontaining330mgofNHS-ActivatedAgaroseSwellvolume:6-7.5uL/mgofdryresin
5columns $125
26200 Pierce NHS-Activated Agarose Slurry 25mLofsettledresininanhydrousacetone
25mL $195
Covalent Coupling of Affinity Ligands to Chromatography Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 27
Thermo Scientific AminoLink Plus Immobilization Kits and Coupling ResinThe simplest and surest method for making an affinity purification resin with antibodies or other proteins.
AminoLinkPlusCouplingResinandImmobilizationKitsuseactivatedbeadedagaroseandarobustcouplingchemistrytoimmobilizeproteinsandotherligandsthroughprimaryamines(–NH2)totheresin.Onceanantibodyorotherligandisimmobilized,thepreparedaffinityresincanbeusedforavarietyofpurificationmethodsinvolvingbatchorcolumnchromatography.Theresinandlinkagearestableinbindingandelutionconditionstypicallyusedinaffinitychromatography,enablingpreparedresintobeusedforatleast10roundsofaffinitypurification.
TheAminoLinkPlusCouplingReactioninvolvesspontaneousformationofSchiffbasebondsbetweenaldehydes(onthesupport)andamines(ontheligand)andtheirsubsequentstabilizationbyincubationwithamildreductant(sodiumcyanoborohydride;seemoredetailedreactionschemeonnextpage).Theentirecouplingreaction,calledreductiveamination,occursinfourtosixhoursinsimplenon-aminebufferssuchasPBS.Couplingefficiencywithantibodiesandtypicalproteinsisgenerallygreaterthan85%,resultingin1to20mgofimmobilizedproteinpermilliliterofagaroseresin.
Highlights:•AminoLink Plus Coupling Resin–aldehyde-activatedcrosslinked 4%beadedagarose• Ideal for antibodies and other proteins–immobilizemolecules viaprimaryamines(–NH2)•Flexible coupling conditions–efficient(>85%)couplingover awiderangeofpH(4–10)andbufferconditions(PBSorother non-aminebufferwithorwithoutorganicsolvent);regular (PBS,pH7.2)andenhanced(borate,pH10)coupling protocolsprovided• Stable, permanent immobilization –couplingreactionresults
instable,leak-resistantsecondaryaminebondbetweenresinandligand
•Better than immobilization to CNBr-activated agarose–bondis morestableanduncharged,resultinginlessnonspecificbinding inaffinitypurificationprocedures•Versatile and reusable–preparedaffinityresinisadaptableto columnandbatchaffinitytechniquesandtheresinisreusable fortypicalapplicationsbasedonproteinbindinginteractions•Convenient kits and product sizes–chooseone-orfive-column kitcontainingcompletesetsofbuffers,reagentsandversatile spin/dripcolumns,orselectbulkresin.Bulkquantitiesare availableformanufacturingapplications
Efficient immobilization of antibodies and other proteins. Percent coupling efficiency of different proteins to 2mL of Thermo Scientific AminoLink Plus Coupling Resin using the pH 7.2 coupling protocol.
ProteinProtein Applied
(mg/mL)Protein Coupled
(mg/mL)
Percent Coupled
ProteinG 4.6 4.0 83
MouseIgG 4.7 4.5 96
RatIgG 4.7 4.4 93
GoatIgG 0.9 0.8 84
HumanIgG 4.8 4.6 97
HumanIgM 0.9 0.8 93
Aga
rose
Bea
d
C
O
H
+ NH2 Protein
Aga
rose
Bea
d
C
O
NH
Protein
Thermo Scientific AminoLink Support immobilization chemistry.
Target: –NH2
Support:Highlycrosslinked4%agarose
Binding capacity:20mgprotein/mLresin
Time: 4hours
ReferencesBeall,A.,et al.(1999).J. Biol. Chem.274(16),11344–11351.Nakasato,Y.R.,et al.(1999).Clin. Chem. 45,2150–2157.Allan,B.B.,et al.(2000).Science289,444–448.Lu,R.,et al.(2000).J. Neurochem.74,320–326
Ordering Information Product #
Description
Pkg. Size
U.S. Price
44894 AminoLink Plus Immobilization Kit Sufficient reagents for preparing five affinity columns. Includes:AminoLinkColumn
PhosphateBufferedSaline(PBS)Citrate-CarbonateBufferQuenchingBufferWashSolutionSodiumCyanoborohydrideSolution(5M)ColumnAccessories
Kit
5x2mL1pack1pack50mL240mL0.5mL
$385
20394 AminoLink Plus Immobilization Trial Kit Sufficient reagents for preparing one affinity column. Includes:AminoLinkColumn
PhosphateBufferedSaline(PBS)Citrate-CarbonateBufferQuenchingBufferWashSolutionSodiumCyanoborohydrideSolution(5M)ColumnAccessories
Kit
1x2mL1pack1pack15mL50mL0.5mL
$105
20475 AminoLink Plus Micro Immobilization Kit Sufficient reagents for 10 coupling reactions using 25–100µg of protein and 20 affinity purifications.Includes:AminoLinkPlusSpinColumns, eachcontaining400μLof25%slurry PhosphateBufferedSaline QuenchingBuffer SodiumCyanoborohydrideSolution WashSolution ElutionBuffer MicrocentrifugeCollectionTubes
Kit10each1pack60mL0.5mL25mL50mL200each
$301
20501 AminoLink Plus Coupling Resin 10mL $105
20505 AminoLink Plus Coupling Resin 50mL $360
28 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Thermo Scientific AminoLink Immobilization Kits and Coupling ResinLinks with primary amines (lysine residues and N-terminus) on proteins, peptides, antigens or antibodies.
AminoLinkCouplingResiniscrosslinked4%beadedagarosethathasbeenactivatedwithaldehydegroups.Proteinsandothermoleculeswithprimaryaminescanbecovalentlyattached(immobilized)toAminoLinkResintomakechromatographycolumnsforuseinaffinitypurification.Thealdehydegroupsformstablesecondaryaminebondswithprimaryaminessuchasexistinthesidechainoflysine(K)residues,whicharegenerallyabundantandreadilyaccessibleinproteins.Onceaproteinisimmobilized,thepreparedaffinityresincanbeusedforavarietyofbatchandcolumnaffinitypurificationmethodsinvolvingbindinginteractionswiththeimmobilizedprotein.Theresinandlinkagearestableinmostbindingandelutionconditionstypicallyusedinaffinitychromatography,enablingpreparedresintobeusedformultipleroundsofaffinitypurificationprocedures.
Highlights:•AminoLink Coupling Resin–aldehyde-activatedcrosslinked 4%beadedagarose• Ideal for antibodies and other proteins–immobilizemolecules viaprimaryamines(–NH2)•Flexible coupling conditions–efficient(>85%)couplingovera widerangeofpH(4–10)andbufferconditions(PBSorother non-aminebufferwithorwithoutorganicsolvent)•Stable, permanent immobilization–couplingreactionresultsin stable,leak-resistantsecondaryaminebondbetweenresin andligand•Better than immobilization to CNBr-activated agarose–bondis morestableanduncharged,resultinginlessnonspecificbinding inaffinitypurificationprocedures•Versatile and reusable–preparedaffinityresinisadaptableto columnandbatchaffinitytechniquesandtheresinisreusable fortypicalapplicationsbasedonproteinbindinginteractions
Aga
rose
Bea
d
C
O
H
+ NH2 Protein
Aga
rose
Bea
d
C
O
NH
Protein
Thermo Scientific AminoLink Support detailed immobilization chemistry.
Target: –NH2
Support:4%agarose
Binding capacity:1-20mgprotein/mLresin
Time: 4hours
Efficient immobilization at a variety of pH values. TheeffectofcouplingbufferpHonpercentcouplingefficiencyof9.58mgofhumanIgGto2mLofThermoScientificAminoLinkResinusingthestandardcouplingprotocol.
pH Coupling Efficiency of 9.58mg Human IgG
4 91.8%
5 92.7%
6 89.1%
7 87.3%
8* 85.3%
9* 94.9%
10* 98.4%
* Schiff-base formation occurs readily at high pH, but reduction to stable secondary amine bond requires subsequent incubation with sodium cyanoborohydride (AminoLink Reductant) at pH 7.2.
ReferencesCheadle,C.,et al.(1994).J. Biol. Chem.269(39),24034–24039.Cofano,F.,et al.(1990).J. Biol. Chem.265(7),4064–4071.DeSilva,B.S.andWilson,G.S.(1995).J. Immunol. Method188,9–19.Rivero-Lezcano,O.M.,et al.(1994).J. Biol. Chem.269(26),17363–17366.Czermak,B.J.,et al.(1999).J. Immunol.162,2321–2325.Assad,F.F.,et al.(2001).J. Cell Biol.152,531–543.Zuk,P.A.andElferink,L.A.(2000).J. Biol. Chem.275(35),26754–26764.6.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20381 AminoLink Coupling Resin 10mL $105
20382 AminoLink Coupling Resin 50mL $380
44890 AminoLink Immobilization Kit Sufficient reagents to prepare five affinity columns. Each column can be used for up to 10 affinity purifications. Includes:AminoLinkColumns
AminoLinkCouplingBufferQuenchingBufferWashSolution,SodiumCyanoborohydrideSolution(5M)ColumnAccessories
Kit
5x2mL250mL50mL240mL0.5mL
$399
20384 AminoLink Immobilization Trial KitIncludes:AminoLinkColumnReagentsandBuffers
TrialKit1x2mL
$106
44892 AminoLink Reductant(Sodiumcyanoborohydride)
2x1g $ 39
Covalent Coupling of Affinity Ligands to Chromatography Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 29
Thermo Scientific UltraLink BiosupportA durable, polyacrylamide resin, activated for efficient coupling of proteins.
UltraLinkBiosupportisadurable,porousresinthatisactivatedtoenableefficientanddirectcovalentimmobilizationofproteinsandotherbiomoleculesthroughtheirprimaryaminesforuseinaffinitypurificationprocedures.
Highlights:•High coupling efficiency and capacity–immobilizesproteins withveryhighefficiencyandcouplingcapacityin1hour•Specific and leak-proof coupling chemistry–reactsspecifically withprimaryamines(–NH2),resultinginamidebondsthatare stableforuseinmanyaffinitypurificationprocedures;coupling reactionhasnoleavinggrouptocontaminatesamples•Easy to use–nopre-swellingorsecondaryreagentsrequired; simplyweightheneededamountofdrysupportandaddthe ligandsolutiontoinitiatecouplingreaction•Flexible coupling conditions–performimmobilizationreaction inanyofavarietyofnon-aminebuffersandpHlevels;coupling ismostefficientinbufferscontainingalyotropicsaltsuchas sodiumcitrate;couplingcompatiblewithorwithoutorganicsolvent•Excellent reusability–preparedaffinityresincanbeusedwith typicalbindingandelutionproceduresformorethan100cycles ofaffinitypurificationwithoutsignificantlossofbindingcapacity•Durable, high-performance resin–porousbeadshavea60μm diameter,canwithstand100psi(6.9bar)andallowforlinear flow-throughratesof3,000cm/hour
H2N+
AmineLigand
Protein
UtraLink Biosupport(Azlactone Ring on Bead)
Amide Bond Formationwith Ring Opening
NHO
NH
O
O
N
O
CH3
CH3
Resi
nB
ead
Resi
nB
ead
Protein
Thermo Scientific UltraLink Biosupport immobilization chemistry.
Target: –NH2
Support:Polyacrylamide/azlactonecopolymer
Binding capacity:>18mgprotein/mLresin
Time: 4hours
References1.Ju,T.,et al.(2002).J. Biol. Chem.277,169–177.2.Ju,T.,et al.(2002).J. Biol. Chem.277,178–186.3.Kornfeld,R.,et al.(1998).J. Biol. Chem. 273,23202–23210.4.Liu,L.A.andEngvall,E.(1999).J. Biol. Chem.274, 38171–38176.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
53110 UltraLink Biosupport (8-10mL) 1.25g $155
53111 UltraLink Biosupport (50mL) 6.25g $539
28388 BupH Citrate-Carbonate Buffer Packs 10packs $ 64
28386 BupH Citrate-MOPS Buffer Packs 10packs $ 58
Thermo Scientific Pierce CDI SupportsCarbonyldiimidazole-activated resins for ligand immobilization.
Highlights:•Reliable coupling chemistry–immobilizationoccursthroughthe reactionofN-nucleophileswith1,1’-carbonyldiimidazolegroups oftheresintoformastable,unchargedN-alkylcarbamatelinkages•Easy-to-use–nosecondaryreagentsneeded;justwashequilibrate resininalkalinecouplingbufferandaddligand;reactionproceeds spontaneously•Stable activation–half-lifeofhydrolysisismuchlongerthan hydroxysuccinimideesteractivations,makingimmobilization reactionssimplertoprepareandcontrol;simplifiesfiltrationand washingbeforeaddingaligandorprotein•Well-defined coupling conditions–reactionismostefficientwith primaryaminesatpH9-11
CDI-Activated Crosslinked 6% Beaded Agarose:•Beadedagarose–themostpopularresinforroutineaffinity purificationmethods•Highlyactivated–atleast50μmol1,1’-carbonyldiimidazole(CDI) groupspermilliliterofresin•Convenientform–suppliedasstabilized,50%slurryinacetone
CDI-Activated Trisacryl® GF-2000:•Trisacrylresin–rigid,polyacrylamidematrixallowsforhighflowrates•Hydrophilicmatrixwithoutchargeeffects–providesforlow nonspecificbinding•Highlyactivated–atleast50μmol1,1’-carbonyldiimidazole(CDI) groupspermilliliterofresin•Convenientform–suppliedasstabilized,50%slurryinacetone
Resi
nB
eadO C
N
N
O
OC
HN
O
H2N
ImidazoleCarbamate
AmineLigand
CarbamateLinkage
Resi
nB
ead
Protein Protein+
CDI immobilization chemistry.
References1.Shenoy,S.K.,et al.(2001).Science294,1307–1313.2.Richardson,R.T.,et al.(2000). J. Biol. Chem.275,30378–30386.3.Tanaka,M,et al.(2005).PLoSBiology3,764–776.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20259 Pierce CDI (6X) Support1,1’-Carbonyldiimidazoleactivatedcrosslinked6%beadedagaroseSupplied:stabilizedinacetoneslurryagarosehydratedparticlesize:45–165μmActivationlevel:>50μmol/mLofresin
10mL $ 85
20377 Pierce CDI Trisacryl Support 50mL $442
30 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Products for Immobilizing Ligands through Sulfhydryl Groups
Thermo Scientific SulfoLink Immobilization Kits and Coupling ResinCovalent immobilization of sulfhydryl-containing peptides or proteins for affinity purification.
SulfoLinkCouplingResinisporous,crosslinked6%beadedagarosethathasbeenactivatedwithiodoacetylgroups.Whenincubatedwithasolutionofpeptideorproteinthatcontainsreducedcysteineresidues,theiodoacetylgroupsreactspecificallyandefficientlywiththeexposedsulfhydryls(–SH)toformcovalentandirrevers-iblethioetherbondsthatpermanentlyattachthepeptideorproteintotheresin.Theresultisacustom-madeaffinityresinforpurifica-tionofantibodies,antigensandothermoleculesofinterest.
Highlights:•Specific conjugation through sulfhydryl (–SH) groups–the iodoacetylgroupsreactspecificallywithsulhydrylstoform irreversiblethioetherbonds•Separate kits optimized for peptides or proteins–kitsinclude optimizedreagentsforpreparingpeptideorproteinsamplesfor efficientimmobilization•Fast –spincolumnsincreaseprotocolspeed;prepareand couplesamplesin2hours(peptides)to3.5hours(proteins)•Flexible coupling conditions–usepH7.5–9.0aqueousbuffers, organicsolvent(e.g.,20%DMSO)ordenaturant(guanidine•HCl), asneededforproteinorpeptidesolubilityduringcouplingreaction•Easy-to-follow instructions–streamLinedprotocolsforsample preparation,immobilization,andaffinitypurification•High capacity–immobilize1–2mgpeptideor2–20mgprotein per2–mLcolumnofSulfoLinkCouplingResin
HS+HN
I
O
HN
S
O
Thermo Scientific SulfoLink Coupling Resin
ThioetherBond
12-atomSpacer
IodoacetylGroup
+ HI
Immobilized Peptide
Reduced SulfhydrylMolecule
Aga
rose
Bea
d
Peptide
Peptide
Aga
rose
Bea
d
Thermo Scientific SulfoLink Support immobilization chemistry.
Target: –SH
Support:6%agaroseorUltraLink®Resin
Binding capacity:20mgprotein/mLresin
Time: 2-3.5hours
Peptide A Calcitonin Peptide0
25
50
75
100
+TCEP
+TCEP
-TCEP-TCEP
Perc
ent
Imm
obili
zed
Improved retention of peptides on Thermo Scientific SulfoLink Resin with TCEP.TCEPeffectivelyreducespeptidestomaximizeimmobilizationefficiency.Twopeptides(PeptideAandhumancalcitoninpeptide)wereincubatedwith25mmTCEPfor30minutesandimmobilizedontoSulfoLinkResinviatheirreducedsulfhydrylgroups.PeptideA’scysteinehadoxidizedduringlong-termstorageandthecalcitoninpeptidecontainedaninternaldisulfidebond.Eachpeptidealsocon-tainedanamine-terminalfluorescentprobebywhichthebindingofthepeptidecouldbemonitoredduringtheimmobilizationandwashsteps. ReferencesGrunwald,R.andMeissner,G.(1995).J. Biol. Chem.270(19),11338–11347.Seubert,P.,et al.(1993).Nature361,260–263.Sukegawa,J.,et al.(1995).J. Biol. Chem.270(26),15702–15706.Wisniewski,J.R.,et al. (1994).J. Biol. Chem.269(46),29261–29264.Sakaguchi,K.,et al.(2000).J. Biol. Chem.275,9278–9283.Tan,M.,et al.(2000).Proc. Natl. Acad. Sci. USA97,109–114.Quill,T.A.,et al.(2001).Proc. Natl. Acad. Sci. USA98,12527–12531.Tokumaru,H.,et al.(2001).Cell 104,421–432.Assad,F.F.,et al.(2001).J. Cell Biol.152,531–543.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
44995 SulfoLink Immobilization Kit for Proteins Sufficient reagents to prepare five affinity columns. Kitcontains:SulfoLinkColumns
SulfoLinkSamplePreparationBufferSulfoLinkCouplingBufferWashSolution2-Mercaptoethylamine•HClL-Cysteine•HClZebaDesaltSpinColumnsPhosphateBufferedSalineColumnsAccessories
Kit
5x2mL7.5mL500mL120mL5x6mg100mg5x5mL1Pack
$ 399
44999 SulfoLink Immobilization Kit for Peptides Sufficient reagents to prepare five affinity columns. Kitcontains:SulfoLinkColumns
SulfoLinkCouplingBufferWashSolutionBond-BreakerTCEPSolutionL-Cysteine•HClPhosphateBufferedSalineColumnAccessories
Kit
5x2mL120mL120mL0.5mL100mg1Pack
$ 399
20325 SulfoLink Immobilization Trial Kit Sufficient reagents to prepare 1 affinity column with a peptide or protein. Kitcontains:SulfoLinkColumn
SulfoLinkSamplePreparationBufferSulfoLinkCouplingBufferWashSolution2-Mercaptoethylamine•HClBond-BreakerTCEPSolutionCysteine•HClZebaDesaltSpinColumnPhosphateBufferedSalineColumnsAccessories
Kit
1x2mL7.5mL120mL25mL6mg0.5mL100mg5mL1Pack
$ 170
20401 SulfoLink Coupling Resin 10mL $ 13520402 SulfoLink Coupling Resin 50mL $ 47420404 SulfoLink Coupling Resin 250mL $1900
Covalent Coupling of Affinity Ligands to Chromatography Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 31
Thermo Scientific UltraLink Iodoacetyl ResinPolyacrylamide resin for coupling sulfhydryl-containing ligands.
UltraLinkIodoacetylResinisadurable,porousresinthatisactivatedtoenableefficientanddirectcovalentimmoblizationofpeptidesandotherligandsthroughtheirsulfhydrylgroups(–SH)foruseinaffinitypurificationprocedures.Thebeadedresinisahydrophiliccopolymerofpolyacrylamideandazlactonehavingarigidpoly-mericstructurewithhighsurfaceareaandporevolume.Eachazlactonegrouphasbeenmodifiedtoforma15-atomspacerarmthatterminatesinaniodoacetylgroup,whichiscapableofreact-ingwithsulfhydrylgroups(e.g.,sidechainofreducedcysteineresidues)tocovalentlyimmobilizepeptideorothersulfhydryl-containingligands.ThebeadstructureandefficientlycouplingchemistryofIodoacetyl-ActivatedUltraLinkSupportresultsinhighproteinbindingcapacity,highlinearflowrates,lownonspecificbindingandoverallsuperiorperformanceinaffinitychromatography.UltraLinkResinsareidealformediumpressureapplicationssuchasFPLC.
Highlights:•High coupling efficiency and capacity–immobilizessulfhydryl- containingproteinsorotherligandswithhighefficiencyand couplingcapacityin1hour•Specific and leak-proof coupling chemistry–reactsspecifically withsulfhydrylgroups(reducedthiols),resultinginthioether bondsthatarestableforuseinmanyaffinitypurificationprocedures•Simple coupling conditions–performimmobilizationreactionin anyofavarietyofbuffers;couplingismostefficientandspecific atpH8.0–8.5;couplingcompatiblewithorwithoutorganicsolvent•Excellent reusability–preparedaffinityresincanbeusedwith typicalbindingandelutionproceduresformanycyclesofaffnity purificationwithoutsignificantlossofbindingcapacity•Durable, high-performance resin–porousbeadshavea60μm diameter,canwithstand100psi(6.9bar)andallowforlinear flow-throughratesof3,000cm/hour
References1.Ju,T.,et al.(2002).J. Biol. Chem.277,169–177.2.Hill,K.,et al.(2000).J. Biol. Chem.275(6),3741–3744.3.Liu,L.A.andEngvall,E.(1999). J. Biol. Chem.274,38171–38176.4.Bicknell,A.B.,et al.(2001).Cell105,903–912.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
53155 UltraLink Iodoacetyl ResinSupport:UltraLinkBiosupport
10mL $188
HS+HN
IO
HN
SO
Iodoacetyl-Activated Thermo Scientific UltraLink Resin
Thioether
Bond15-atomSpacer
IodoacetylGroup
+ HI
Immobilized PeptideReduced SulfhydrylMolecule
Ther
mo
Scie
ntifi
c U
ltraL
ink
Bea
d
Ther
mo
Scie
ntifi
c U
ltraL
ink
Bea
d
Peptide Peptide
Thermo Scientific UtraLink Iodoacetyl immobilization chemistry.
Thermo Scientific UltraLink Iodoacetyl Micro Peptide Coupling KitEasily prepare a small-scale affinity column with sulfhydryl- containing peptides.
TheMicroPeptideCouplingKitisamicrocentrifugespincolumnkitforimmobilizingsmallamounts(25–250μg)ofsulfhydryl-containingpeptides(e.g.,cysteine-terminatedpeptides)ontoabeadedporousresintocreateasmall,reusableaffinitycolumn.Thecouplingandaffinitypurificationproceduresareoptimizedforsmallsamplevolumes(200–300μL).Washandelutionstepsareachievedrapidlyandefficientlywiththeconvenientmicrocentrifugespincolumns.Eachkitcontainssufficientreagentsfor10couplingreactionsand20affinitypurifications.Thekitisidealforimmobilizingpeptideantigensthatcontainingaterminalcysteineresidueforuseinpurifyingspecificantibodiesfromsmallserum,ascitesorculturesupernatantsamples.
Highlights:•Optimized for small scale–idealforcouplingsmallamounts (25–250μg)ofsulfhydryl-containingpeptideorproteinfor purificationofspecificantibodiesfromcrudeserum•High-performance affinity resin–usesdurable,polyacrylamide- basedUltraLinkIodoacetylResinforspecificreactionto sulfhydrylgroups•Efficient coupling chemistry–immobilizationefficiency>85% (asmeasuredwith1hourreactionusinginsulin,calcitoninand osteocalcinpeptides)•Fast and easy to use–performwashandelutionstepsusing amicrocentrifuge•Reusable–usepreparedpeptideresinseveraltimeswithno significantlossofcapacity
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20485 Micro Peptide Coupling Kit Sufficient for coupling 10 sulfhydryl-containing peptides or proteins and perform 20 affinity purifications. Kitcontents:UltraLinkIodoacetylSpinColumns,0.1mL
CouplingBufferL-Cysteine-HClWashSolutionPBSPack(makes500mL)IgGElutionBufferCollectionTubes
Kit
10columns100mL100mg25mL1pack50mL200tubes
$357
32 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Thermo Scientific Disulfide Reducing AgentsReduce disulfide bonds to produce sulfhydryl groups for immobilization on SulfoLink or UltraLink Resins.
Freesulfhydrylsarerequiredforimmobilizationontosulfhydryl-reactiveaffinitysupports.Cysteinesinproteinsandpeptidesusuallyexistascystines(disulfidebridges)andmustbereducedtoexposesulfhydrylsforcoupling.Reductioncanbeaccomplishedwithfreeorimmobilizedreducingagents.Freereducingagentsareefficientinreducingalldisulfidesinproteins,includingthoseburiedinthetertiarystructure,buttheymustberemovedfromthereducedsamplewithadesaltingcolumnbeforecouplingtothesupport.Immobilizedreducingagentsenablereductionofdisulfidesandsimpleremovalofthereducedsamplefromthereducingagent.Thisisespeciallyhelpfulwhenreducingpeptideswhosesmallsizepreventsthemfrombeingeffectivelydesalted.
PHO
O
OHO
OH
O
Cl–
TCEP•HClMW 286.65
H+
HSNH3 Cl–
2-Mercaptoethylamine•HCIMW 113.61
SHHS
OH
OH
DTTMW 154.25
+
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20408 2-Mercaptoethylamine•HCl 6x6mg $135
20290 DTT, Cleland’s Reagent(Dithiothreitol)
5g $105
20291 Dithiothreitol (DTT) in No-Weigh™ Format7.7mgDTT/tube.Makes100μLof0.5MDTT.
48tubes $125
20490 TCEP•HCl(Tris[2-carboxyethyl]phosphinehydrochloride)
1g $ 50
20491 TCEP•HCl 10g $299
77720 Bond-Breaker TCEP Solution, Neutral pH 5mL $108
77712 Immobilized TCEP Disulfide Reducing Gel 5mL $118
Thermo Scientific Ellman’s Reagent and Sulfhydryl Addition KitMeasure and add free sulfhydryls to ensure success of cysteine-targeted immobilization.
Ellman’sReagent,alsocalledDTNB,isaversatilewater-solublecompoundforquantifyingfreesulfhydrylgroupsinsolution.Asolutionofthiscompoundproducesameasurableyellow-coloredproductwhenitreactswithsulfhydryls(–SHgroups).Bytestinganunknownsample,suchasapeptidehavingaterminalcysteineresidue,comparedtoastandardcurvemadewithknownamountsoffree,reducedcysteine(Product#44889),availabilityofreducedsulfhydrylsinthesamplecanbedetermined.
TheSulfhydrylAdditionKitprovidestheessentialreagentsandprocedureforcreatingnewsulfhydrylgroupsonaproteinorothermoleculethatcontainsavailableprimaryamines(–NH2).ThekitusesSATAreagent,whichformscovalentbondstoprimaryamines.Theresultisadditionofastable(capped)sulfhydrylgroup,whichcanlaterbeexposedbygentletreatmentwithhydroxylamine,makingthemoleculereadyforconjugationtoSulfoLinkCouplingResin,UltraLinkIodoacetylResinorothersulfhydryl-reactiveimmobilizationmethod.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
23460 Sulfhydryl Addition KitAdds free sulfhydryl groups to proteins.Includes:SATA Hydroxylamine•HCl 10XConjugationBufferStock PhosphateBufferedSalinePack Dimethylformamide(DMF) DextranDesaltingColumn ColumnExtender Ellman’sReagent(DTNB) Cysteine•HClH2O
Kit2mg5mg20mL1pack1mL1x5mL12mg20mg
$223
22582 Ellman’s Reagent(5,5’-Dithio-bis-[2-nitrobenzoicacid])
5g $ 74
44889 Cysteine•HCl 5g $ 45
Covalent Coupling of Affinity Ligands to Chromatography Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 33
Products for Immobilizing Ligands through Carbonyl Groups
Thermo Scientific CarboLink Immobilization Kit and Coupling ResinsImmobilize polyclonal antibodies and other glycoproteins through carbohydrate groups.
CarboLinkCouplingResinandKitsprovideforcovalentimmobiliza-tionofglycoproteinsandothercarbohydrate-containingmoleculestobeadedagarose(orpolyacrylamideUltraLinkSupport)foruseinaffinitypurificationprocedures.Carbohydratemoietiesinglyco-proteinscontaincommonsugarswhosecis-diolgroupsareeasilyoxidizedwithsodiummeta-periodate(includedintheCarboLinkKit)toyieldaldehydes.WhenincubatedwiththeCarboLinkResin,thesealdehydegroupsreactspontaneouslywiththehydrazidegroupoftheactivatedresintoformstable,covalentbonds.Theimmobilizationstrategyisespeciallyusefulforglycoproteins,suchaspolyclonalantibodies,becauseitallowsattachmentofthemoleculeatdomainsthatwillnotinterferewithbindingsitesthatarecriticalfortheintendedaffinitypurification.Onceamoleculeiscoupled,thepreparedaffinityresincanbeusedmultipletimesintypicalproteinaffinitypurificationprocedures.
Highlights:•CarboLink Coupling Resin–hydrazide-activatedcrosslinked 6%beadedagarose(orhydrazide-activatedUltraLinkSupport, abeaded,polyacrylamideresin)•Efficient immobilization–couple1–5mgofoxidizedpolyclonal antibodyorotherglycoproteinpermilliliterofresin(CarboLink Resincontainsgreaterthan14μmolhydrazidegroupspermilliliter)•Stable linkage–resonancestructureofthehydrazonebonds aresufficientlystabletoallowmultipleroundsofaffinity purificationwithonebatchofpreparedresin;nostabilizing reductantrequired•Flexible and gentle coupling conditions–immobilizationreaction completedinsimplebuffers(PBSorothernon-aminebufferwith orwithoutorganicsolvent)atnear-neutralpH• Ideal for polyclonal antibodies–immobilizesIgGthrough carbohydratesintheFcregion,sobothantigenbindingsitesare freetointeractwiththeantigeninthemobilephase•Effective for any molecule with oxidizable sugars–firststepis oxidationofthesugargroups,whichallowsthecis-diolsofthe IgGtobetransformedintoreactivealdehydemoieties;these aldehydesthencombinewithhydrazidegroupsonthematrixto formstable,leak-resistantlinkages•Convenient kits and product sizes–chooseone-orfive-column kitcontainingcompletesetsofbuffers,oxidizingreagentand versatilespin/dripcolumnscontainingthebeadedagaroseresin; orchoosethepolyacrylamide-basedUltraLinkSupport.Bulk quantitiesareavailableformanufacturingapplications
Aga
rose
Bea
d
Aga
rose
Bea
d
NH
NH2
O
NH
NO
Thermo Scientific CarboLink Resin
23-atomSpacer
HydrazideGroup
Antibody with CarbohydrateGroups Oxidized to Form
Aldehyde Groups
Immobilized Glycoprotein
Oxidized Glycoprotein
HydrazoneBond
H
O
Thermo Scientific CarboLink Support immobilization chemistry.
Target: –CHO
Support:6%agarose
Binding capacity:5mgprotein/mLresin
Time: 8hours
References1.Kumar,P.G.,et al.(2001).J. Biol. Chem.276,41357–41364.2.Strakova,Z.,et al.(1997).Mol. Pharmacol.51,217–224.3.Brown,M.A.,et al.(2000).J. Biol. Chem.275,19795–19802.4.Butko,P.,et al.(1999).J. Immunol.163,2761–2768.5.Sequra,M.,et al.(1999). Infect. Immun.67(9),4646
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20355 CarboLink Immobilization Trial KitKitcontains:CarboLinkColumn
CarboLinkCouplingBufferCarboLinkWashSolutionSodiummetaperiodateZebaDesaltingColumn
Kit1x2mL60mL15mL1x5mg1x5mL
$155
44910 CarboLink Immobilization KitKitcontains:CarboLinkColumns
CarboLinkCouplingBufferCarboLinkWashSolutionSodiummetaperiodateZebaDesaltingColumns
Kit5x2mL250mL100mL5x5mg5x5mL
$454
20391 CarboLink Coupling Resin 10mL $11353149 UltraLink Hydrazide Resin 10mL $18620504 Sodium meta-Periodate 25g $ 29
34 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Products for Immobilizing Ligands through Carboxyl Groups
Thermo Scientific CarboxyLink Immobilization Kits and Coupling ResinsImmobilize peptides via carboxyl groups to create an affinity column.
CarboxyLinkCouplingResinandKitsprovideforcovalentimmobil-izationofpeptidesorothercarboxyl-containing(–COOH)moleculestoaporous,beadedresinforuseinaffinitypurificationprocedures.CarboxyLinkResiniscrosslinkedbeadedagarose(orpolyacryl-amideUltraLinkSupport)thathasbeenactivatedwithdiamino-dipropylamine(DADPA)tocontainlongspacerarms,eachwithaprimaryamineattheend.WhenincubatedwiththeresinandthecarbodiimidecrosslinkerEDC(includedintheCarboxyLinkImmobilizationKit),carboxyl-containingmoleculesbecomeper-manentlyattachedtothesupportbystableamidebonds.Onceamoleculeiscoupled,thepreparedaffinitycolumncanbeusedmultipletimesintypicalproteinaffinitypurificationprocedures.CarboxyLinkCouplingResinscanalsobeusedtoimmobilizeotherkindsofmoleculesusingalternativeamine-reactivecrosslinkingchemistries.
Highlights:•CarboxyLink Coupling Resin–DADPA-activatedcrosslinked 4%beadedagarose(orDADPA-activatedUltraLinkSupport, abeadedpolyacrylamideresin)•Efficient immobilization–couple1–2mgofpeptidepermilliliter ofresin(CarboxyLinkAgaroseResinactivatedwithgreaterthan 16μmolaminemilliliterofresin;DADPAonUltraLinkSupport activatedwithgreaterthan40μmolaminemilliliterofresin)•Stable linkage–immoblizationresultsincovalentattachmentof carboxylgroupsbyamidebonds,allowingformultipleroundsof affinitypurificationwithonebatchofpreparedresin•Flexible and gentle coupling conditions–immobilizationreaction completedinsimpleMESorothernon-amineandnon-carboxyl, near-neutralbuffer,withorwithoutorganicsolvent.• Ideal for unmodified peptides–immobilizespeptideswith highcapacityandvariousorientationswithoutsteric hindrance,allowingforeffectiveuseinaffinitypurificationof specificantibodies•Convenient kits and product sizes–choosefive-columnkitswith completesetsofbuffers,crosslinkerandversatilespin/drip columnscontainingeithertypeofresin(agaroseor polyacrylamide)orchoosestand-aloneresinforotheruses
HN
HN NH2
Immobilized DADPA(Thermo Scientific CarboxyLink Resin)
HN
HN
HN
+O
HO
O
EDC Crosslinker
Carboxyl Ligand(e.g., peptide C-terminus)
Covalently Immobilized Ligand(attached by amide bond and long spacer arm)
Aga
rose
Bea
d
Peptide
Peptide
Aga
rose
Bea
d
Thermo Scientific CarboxyLink Support immobilization chemistry.
Target: –COOH
Support:4%agaroseorUltraLink®Resin
Binding capacity:5mgprotein/mLresin
Time: 4hours
ReferenceYoo,B.C.,et al. (2002).J. Biol. Chem.277,15325–15332.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
44899 CarboxyLink Immobilization KitKitcontains:CarboxyLinkColumns(DADPAagarose)
EDCCrosslinkerCouplingBuffer(MES-bufferedSaline)WashSolution(1MNaCl)ColumnAccessories
Kit
5x2mL5x60mg500mL120mL
$393
20266 CarboxyLink Coupling ResinSupport:Crosslinked4%beadedagaroseLoading:16–20μmolavailableaminogroups/mLofresin
25mL $145
53154 CarboxyLink Immobilization Kit with UltraLink SupportKitcontains:DADPAUltraLinkColumns
EDCCrosslinkerCouplingBuffer(MES-bufferedSaline)WashSolution(1MNaCl)ColumnAccessories
Kit
5x2mL5x60mg500mL120mL
$432
22980 EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride
5g $ 83
28390 BupH MES Buffered Saline Packs 10pack $112
Covalent Coupling of Affinity Ligands to Chromatography Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 35
Antibody immobilization: choosing the best Thermo Scientific Support.
AminoLink Plus Coupling Resin
UltraLink Biosupport
CarboLink Coupling Resins
SulfoLink Coupling Resins
Crosslink IP Kits See page 41
Monoclonal Antibodies Advantages:•Goodchoicewhen
onlysmallamountsofantibodyareavailable
•CoupleoverabroadpHrange
•Goodcouplingefficiency
Disadvantages:•ReductionofSchiff’sbase
withsodiumcyanoborohy-dridemayadverselyaffectmonoclonals
•Someantibodiesmaybecoupledthroughantigen-bindingsite
Advantages:•Goodchoiceifantibody
canwithstand1.0Msodiumcitrateorsulfate
•Highcapacity•Fast,efficientcoupling•Good,forlarge-scaleor
fast-flowapplications
Disadvantages:•Someantibodiesmaybe
coupledthroughantigen-bindingsite
•Someantibodiesmaypre-cipitateinhigh-saltbuffer
Advantages:•Correctlyorientsantibody•Antibodymustbeable
towithstandoxidationconditions
•Goodforantibodieswithlowavidityforantigen
Disadvantages:•Notallmonoclonalshave
carbohydrateaccessibleforcoupling
•Conditionsnecessaryforcouplingmayadverselyaffectsomemonoclonals
Advantages:•Goodchoicefor
antibodiesthathaveextremelyhighavidityfortheirantigen
•Allowsforgentleelutionconditions
Disadvantages:•Mustfirstreduceantibody
priortocoupling•Notgoodforantibodies
withlowaffinityfortheirantigens
Advantages:•Allowsforcorrect
orientationofantibodies•Gentlecouplingconditions•ProteinA/Gwillbind
mostantibodies
Disadvantages:•Ifpurifyingantigenfrom
serum,antibodiesmaybindtoProteinA/Gandco-purifywithantigen
•Crosslinkingresultsinsomelossofantibodyactivity
Polyclonal Antibodies Advantages:•Excellentcoupling
efficiency•Goodantigenrecovery
Disadvantages:•Someantibodiesmay
becoupledthroughantigen-bindingsite
Advantages:•Goodchoicefor
large-scaleorfast-flowapplications
•Highcapacity•Fast,efficientcoupling
Disadvantages:•Someantibodiesmay
becoupledthroughantigen-bindingsite
•Someantibodiesmayprecipitateinhigh-saltbuffer
Advantages:•Correctlyorientsantibody•Antibodymustbeable
towithstandoxidationconditions
•Goodforantibodieswithlowavidityforantigen
Disadvantages:•Conditionsnecessaryfor
couplingmayadverselyaffectsomeantibodies
Advantages:•Goodchoiceforanti-
bodiesthathaveavidityfortheirantigen
•Allowsforgentleelutionconditions
Disadvantages:•Mustfirstreduceantibody
priortocoupling•Notgoodforantibodies
withlowaffinityfortheirantigens
Advantages:•Allowsforcorrect
orientationofantibodies•Gentlecoupleconditions•ProteinA/Gwillbind
mostantibodies
Disadvantages:•Ifpurifyingantigenfrom
serum,antibodiesmaybindtoProteinA/Gandco-purifywithantigen
•Crosslinkingresultsinsomelossofantibodyactivity
High-Activity Antibodies Advantages:•Immobilizationofreduced
antibodyallowsforgentlerelutionconditions
Low-Activity Antibodies Advantages:•Correctlyorientsantibody
Disadvantages:•Conditionsnecessaryfor
couplingmayadverselyaffectsomemonoclonals
Advantages:•Allowsforcorrect
orientationofantibodies•Gentlecoupleconditions•ProteinA/Gwillbind
mostantibodies
Disadvantages:•Ifpurifyingantigenfrom
serum,antibodiesmaybindtoProteinA/Gandco-purifywithantigen
•Crosslinkingresultsinsomelossofantibodyactivity
36 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Immunoprecipitation
The topic of co-immunoprecipitation (co-IP) is best preceded by a discussion of immunoprecipitation (IP) to help frame an under-standing of the principles involved.
IPisoneofthemostwidelyusedmethodsforantigendetectionandpurification.AnimportantcharacteristicofIPreactionsistheirpotentialtodelivernotonlythetargetprotein,butalsoothermacromoleculesthatinteractwiththetarget.
The IP PrincipleTheprincipleofanIPisverysimple(Figure1).Anantibody(monoclonalorpolyclonal)againstaspecifictargetantigenisallowedtoformanimmunecomplexwiththattargetinasample,suchasacelllysate.TheimmunecomplexisthencapturedonasolidsupporttowhicheitherProteinAorProteinGhasbeenimmobilized(ProteinAorProteinGbindstotheantibody,whichisboundtoitsantigen).Theprocessofcapturingthiscomplexfromthesolutionisreferredtoasprecipitation.Anyproteinsnot“precipitated”bytheimmobilizedProteinAorProteinGsupportarewashedaway.Finally,componentsoftheboundimmunecomplex(bothantigenandantibody)areelutedfromthesupportandanalyzedbySDS-PAGE(gelelectrophoresis),oftenfollowedbyWesternblotdetectiontoverifytheidentityoftheantigen.
Coupled Antibody
Spin and wash
Cell Lysate or Protein MixtureIncubation with
Antibody-coupled Resin
Antigen
Elute
Analyze
Figure 1. Summary of a traditional immunoprecipitation procedure.
Traditionalimmunoprecipitationinvolvesthefollowingsteps:
1.Formtheantigen-antibodycomplex(immunecomplex)by incubatingspecificantibodywiththeantigen-containingsample for1hourtoseveralhours.2.CapturetheimmunecomplextoProteinAorProteinGagarose beadsbyincubationfor0.5-2hours.3.Removeanynon-boundprotein(non-immunecomplexsample components)fromtheprecipitatedcomplexbywashingbeads withadditionalsamplebuffer.4.BoilbeadsinreducingSDS-PAGEsampleloadingbuffer.5.Recoverelutedsampleinloadingbufferandanalyzeby SDS-PAGE.6.PerformWesternblotanalysis,probingwithantigen- specificantibody.
Co-IP vs. IPCo-IPisapopulartechniqueforproteininteractiondiscovery.Co-IPisconductedinessentiallythesamemannerasIP(Figure2).However,inaco-IPthetargetantigenprecipitatedbytheantibody“co-precipitates”abindingpartner/proteincomplexfromalysate;i.e.,theinteractingproteinisboundtothetargetantigen,whichbecomesboundbytheantibodythatbecomescapturedontheProteinAorProteinGresin.Theassumptionusuallymadewhenassociatedproteinsareco-precipitatedisthattheseproteinsarerelatedtothefunctionofthetargetantigenatthecellularlevel.Thisisonlyanassumption,however,thatissubjecttofurtherverification.
Coupled AntibodySpin and wash
Cell Lysate or Protein MixtureIncubation with
Antibody-coupled Resin
Antigen
Protein Interactingwith Antigen
Elute
Analyze
Figure 2. Summary of a traditional co-immunoprecipitation procedure.
IP/Co-IP
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 37
Traditional Methods vs. Thermo Scientific Pierce Innovations for Co-IP
Problems with Traditional Co-IP MethodsThetraditionalco-IPprotocolhascertaindeficienciesrelatingtothefundamentalformatoftheassay,theantibodyandassociatedchemistry.OneofthemostcommonlyencounteredproblemswiththetraditionalIPandco-IPapproachisinterferencefromantibodybandsingelanalysis.Inthosecasesinwhichseveralproteinsmaybeco-precipitatedwiththetarget,presenceoftheco-elutedantibodyheavyandlightchains(25and50kDabandsinreducingSDS-polyacrylamidegel)inthepreparationcanobscuretheresults.Theidealsituationwouldbetoconducttheco-IPwithoutcontaminationoftheelutedantigenwithantibody.Withthispotentialinterferenceeliminated,onlytheco-precipitatedproteinswillbepresentanddetectedonagel.ThisandothershortcomingsofthetraditionalprotocolandoursolutionsaresummarizedinTable1.
Table 1. Comparison of traditional Co-IP and Thermo Scientific Pierce Co-IP Products.
Traditional Co-IP Problems Thermo Scientific Pierce Product Solutions
Batch processing of the precipitated complex in a single tube:resultsininefficientwashingofnon-boundproteinsfromthesupportandinresinlossduetodecantingwashbufferfromtubeviaapipette,whichlowersproteinyields
Spin cup or spin tube processing:dedicatedIPandco-IPkitsthatcontainspin-cuporspintubedevicesthatincreasewashingefficiencyoffermoreeffectiveelutionofantigenandassociatedproteinandeliminateresinloss,yieldingsignificantlyhigherproteinrecoveriesandmoreconsistentresults
Antibody fragment interference:co-elutionofantibodyfragmentswithantigenoftenresultsinbandsinterferingwithdetectionofanyco-precipitatedproteinsonSDS-PAGE
Antibody immobilization:chemistriesdesignedtoimmobilizetheantibodytothesupport,therebyallowingelutionofonlythetargetandanyassociatedproteinsinaco-IPcomplex
Eliminate detection of antibody used for immunoprecipitation:contaminatingantibodyfragmentsbecomedenaturedduringSDS-PAGE.Incontrast,theprimaryantibodyusedduringWesternblottingisstillnative.UseCleanBlotHRPDetectionReagent(Product#21230)toonlydetectnativeantibodyandnotcontaminants.
Antibody sacrificed:asaconsequenceofharshelutionconditions,thetargetantibodyisdestroyed;antibodylossbywayoftheprotocolcanbecostly
Antibody re-used:immobilizationchemistryandmildelutionconditionsforthetargetandassociatedproteinsallowtheimmobilizedantibodytobere-equilibratedandrecycledseveraltimesintheco-IPprotocol
Approaches to Co-IP Free of Antibody Interference
Three approaches have been incorporated into several products targeted to IP and co-IP applications.
Activated Support for Antibody Immobilization — Direct StrategyInthisapproach,anantibodyisimmobilizeddirectlythroughitssurfaceaminegroups(contributedprimarilybythesidechainepsilon-aminogroupoflysine)toahigh-capacityaldehyde-activatedbeadedagarosesupport(ThermoScientificAminoLinkPlusCouplingResin).ThesupportformsaSchiff’sbasewiththeseavailableaminesthatisreducedtoformstablesecondaryaminebondsduringtheimmobilizationprocess.Thewiderangeofcouplingconditionsthatcanbeusedwiththissupportmakeitidealformaintainingbiologicalbindingactivitycriticaltothesuccessfulexecutionofaco-IPexperiment.WhenusingtheDirectStrategy,theantibodysourceshouldbefreeofcarrierprotein,whichcanalsobeimmobilized.ProductssuchasThermoScientificMelonGelIgGPurificationKits(Product#45206)caneasilyclean-upanantibodytoremovecarrierproteins.Thedirectimmobilizationofantibodiesisnotspeciesdependent,whichallowsfortheuseonnon-traditionalantibodysourcesthatdon’tbindglobulinbindingproteins(ProteinAorProteinG).ThermoScientificPierceCo-ImmunoprecipitationKits(Product#26149)andThermoScientificPierceDirectIPKit(Product#26148)usethisdirectimmobilizationapproach.Forco-IPapplica-tions,theflexibility,simplicityanddurabilityofthedirectmethodasanantibody-couplingstrategymakesitthemethodofchoicefordeliveringresultsfreefromantibodyinterference.
Antibody Orientation and Immobilization — Indirect StrategyThisstrategytakesadvantageofthebindingcharacteristicsofthetraditionalProteinAorProteinGagarosecombinedwithchemicalcrosslinkingtocovalentlylinktheantibodytothesupport.ProteinAandProteinGbindIgGclassantibodiesthroughtheFcregionthatischaracterizedprimarilybydimerizedheavychainmodifiedbycarbohydrate.Fcregionbindingnaturallyorientstheantigen-bindingdomainsoftheantibody(Fab)awayfromthesupport,makingthemavailableforbindingtotheirrespectivetargetantigen.TheIndirectStrategyiscompatiblewithantibodysamplesthatcontaincarrierproteins,providedthecarrierproteinsarewashedawaypriortocrosslinking.Toensurethattheantibodyremainsonthesupportduringtherequisiteantigenbinding,washandelutionstepsoftheprotocol,thisboundandorientedantibodyischemi-callycrosslinkedtotheProteinAorProteinGwiththebifunctionalreagentdisuccinimidylsuberate(DSS).ThermoScientificPierceCrosslinkIPKit(Product#26147)incorporatesthisstrategy.
38 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Use of the Streptavidin:Biotin InteractionThisdirectcouplingapproachincorporatesthebindingassociationbetweenstreptavidinandbiotin.StreptavidinimmobilizedtobeadedagaroseresinorcoatedinmicroplatewellsprovidesanalternativeIPorco-IPstrategyforobtainingresultsfreefromantibodyinter-ference.Biotinylatedantibodyisboundverystronglytoeachmatrixandisnotelutedwhenmildconditionsareusedtoreleasethetargetantigen.TheIP/co-IPisconductedbyincubatingthesamplewiththebiotinylatedantibody-loadedmatrix.Elutionofthetargetantigenandanyinteractingproteinsisperformedfreeofantibodycontamination.ThermoScientificPierceStreptavidinAgaroseResin(e.g.,Product#20347)andkitproductsthatusethissupport,aswellastheThermoScientificPierceStreptavidinCoatedPlateIPKit(Product#45360),providehigh-capacitybiotin-bindingmatricessuitableforIPandco-IPapplications.
Optimization Parameters in IP and Co-IP
Classical Immune Complex Formation vs. Pre-Binding of AntibodyAchangeinprotocolfromtheclassicalimmunecomplexprecipitationisnecessarywhenusingimmobilizedantibodyintheco-IPmethod.Inthetraditionalco-IPprotocol,theimmunecomplex(antigen:antibody)isformedinsolutionbefore“precipitating”itwiththeimmobilizedProteinAorProteinGmatrix.Whenusingimmobilizedantibody,theimmunecomplexisformeddirectlyontheantibody-coupledmatrixbyincubationoftheantigen-containingsamplewiththematrix.Formationoftheimmunecomplex(thetargetantigenandanytarget-associatedprotein)anditsprecipitationoccursinonestep.
Intheimmobilizedformat,theantibodyisallowedtoincubatewiththelysate.Thematrixiswashedusingaspincupformatandtheboundproteinelutedforanalysis.Thetargetantigenandco-IPcomplexisrecoveredfreeofantibodyorantibodyfragmentcontamination,andtheantibodyisretainedinanactiveformonthesupporttobeusedinanotherco-IPcycle.
Ourresearchindicatesthatpre-bindingor-couplingtheantibodytothesupportmatrixconsistentlyresultsinthecaptureofmoretargetantigen,evenincoatedplateIPproceduresthatdonotrequireit.ThisapproachisrecommendedfortheThermoScientificPierceCoatedPlateIPKitsthatuse96-wellmicroplatescoatedwithstreptavidinorProteinA/G(Products#45360,45350,respectively).Antibodyisboundtotheplatewellspriortotheprescribedincubationwithalysatesample.Unboundproteiniseasilywashedfromthewellspriortotheelutionofthetargetandanyco-precipitatedproteins.
Evaluating a Co-IP-Captured Interaction
Intheirreviewofproteininteractions,PhizickyandFields(seeReferenceslistedbelow)presentadiscussionoftheissuestoconsiderinvalidatingasuspectedinteractionobtainedbyaco-IPexperiment.Ultimately,thefollowingquestionmustbeanswered:Doestheinteractiondetectedbyco-IPoccurin vivo,andwhatsignificancedoesithaveatthecellularlevel?AsummaryofthePhizickyandFieldsapproachtoverificationofco-IPdatafollows.
Confirm that the co-precipitated protein is obtained only by antibody against the targetUsemonoclonalantibodiesintheco-IPprotocol.Whenonlyapolyclonalantibodyisavailable,pre-treatmentoftheantibodywithsampledevoidoftheprimarytarget(baitprotein)mayberequiredtoassurethatthepolyclonalantibodydoesnotcontainclonesorcontaminantsthatbindpreyprotein(s)directly.Pre-adsorptiontoextractsdevoidoftargetorpre-purificationofpolyclonalIPantibodiesagainstanaffinitycolumncontainingpuretargetantigensafeguardsagainstafalse-positiveco-IP.
Conclude that antibody against the target antigen does not itself recognize the co-precipitated protein(s)Useindependentlyderivedantibodiesthathavedemonstratedspecificitiesagainstdifferentepitopesonthetargetprotein.Theiruseservesasverificationthatthetarget(bait)-directedantibodieshavenoaffinityforthetarget-associatedpreyproteinsrecoveredduringtheco-IP.Alternatively,anantibodyagainsttheco-precipitatedproteincanbeusedtoco-IPthesamecomplex.
Determine if the interaction is direct or indirectIstheinteractionmediatedthroughathird-partyproteinthatcontactsbothtargetandco-precipitatedprotein?Immunologicalandothermoresophisticatedmethodssuchasmassspectrometrymaybenecessarytoanswerthisquestion.
Determine that the interaction takes place in the cell and not as a consequence of cell lysis Suggestedapproacheshereinvolveco-localizationstudiesandsite-specificmutagenesisgivingrisetomutantsthatperturbthebindingprocess.
ReferencesAdams,P.D.,et al.(2002).Identificationofassociatedproteinsbyco-immunoprecipitation,In Protein-Protein Interactions – A Molecular Cloning Manual.Golemis,E.,Ed.,ColdSpringHarborLaboratoryPress,pp59-74.
Liebler,D.C.(2002).Identifyingprotein-proteininteractionsandproteincomplexes.In Introduction to Proteomics, Tools for the New Biology,HumanaPress,pp.151-165.(Product#20061)
Phizicky,E.M.andFields,S.(1995).Protein-proteininteractions:methodsfordetectionandanalysis.Microbiological Reviews(Mar.),pp.94-123.
IP/Co-IP
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 39
IP and Co-IP Kits
TheThermoScientificPierceClassicandCrosslinkIPKitsareidealwhenusingantibodiesthatbindtoProteinAorProteinG.TheDirectandCrosslinkIPKitsarehighlyeffectiveforeliminatingco-elutionofIgGheavyandlightchainwiththeantigen,whichinterfereswithdownstreamapplicationssuchasmassspectrometryanalysisorproteinsequencing.
Traditionalco-IPmethodsresultindetectionoftheantibodywiththetargetproteins.Becausetheantibodyheavyandlightchainsmayco-migratewithoneoftherelevantbands,importantresultscanbemasked.ThePierceCo-IPKitcircumventsissueswithco-migrationofantibodychainswithtargetproteinsbyretain-ingtheantibodyontheresin.Thenewkitisoptimizedforusingsmalleramountsofsampleandoffersasinglelysis/washbuffereliminatingtheneedforaseparatelysisreagent.
All four IP Kits Highlights:•Requireminimalantibody(2-10μg)•Arehighlyeffectiveandefficientincapturingantigens•UseoptimizedprotocolsandbuffersforefficientIPsand
antigenelution•Usecommonlysis/binding/washbuffer•Includespincolumnsandcollectiontubesthatshortenthe
protocolbyminimizinghandlingandmixing•Useanewelutionbufferthatprovidesmilderandlessdenaturing
recoveryofantibody:antigencomplexes•Arecompatiblewithspecializeddownstreamapplications;e.g.,
massspectrometry,enzymeassaysandantibodyproduction•Areabletoscaleupasneededusingourflexibleprotocol
Comparison of immunoprecipitation methods. Thefollowingtablecomparesthekeyfeaturesoftraditional“do-it-yourself”immunoprecipitationtechniquestotheThermoScientificPierceIPKits.Considerationofthesefeaturescanhelptodeterminewhichmethodismostappropriatefortheavailablereagentsanddownstreamapplication.
Feature
Traditional IP method
Pierce Classic IP Kit
Pierce Crosslink IP Kit
Pierce Direct IP Kit
Pierce Co-IP Kit
Useshighbindingcapacityresin Variable1 Yes(ProteinA/GPlus)
Yes(ProteinA/GPlus)
Yes(AminoLinkPlus)
Yes(AminoLinkPlus)
Crosslinkermediatedimmobilization No No Yes(DSS)
No No
Requirespurifiedantibodyinamine-freeandprotein-freestoragesolution
No No No Yes Yes
Antibodyiscovalentlyattachedtoagaroseresin No No Yes Yes Yes
Antibodyisoriented Yes Yes Yes No No
Antibodyeluteswithantigen Yes Yes No No No
Antigenrecoverymethod Boilingw/SDS(LowpH)
LowpHelution(Boilingw/SDS)
LowpHelution
LowpHelution
LowpHelution
Relativeantigenrecovery2 Variable Highest Medium High High
Immobilizedantibodycanbereused No No Possible3 Possible3 Possible3
Co-purificationofinteractingproteins Possible Possible Possible Optimal
1 Commercially available resins vary in binding capacity and performance for IP assays.2 Antigen yield depends on the activity of the antibody, specific binding conditions and the immobilization method used.3 It is possible to reuse the prepared antibody affinity resin if the antibody remains functional following low pH elution.
40 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Comparison of three different Thermo Scientific Pierce IP Kits. Immunoprecipitationswereperformedaccordingtotheproductinstructionsusing10μgofaffinity-purifiedgoatanti-GFPantibodyandthePierceDirect,ClassicandCrosslinkIPKits.ThecelllysatewaspreparedusingIPLysis/WashBufferandpre-clearedusingthePierceControlAgaroseResinsuppliedinthekits.Theimmunecomplexwasformedbyincubatingtheantibody,resinandlysateovernight.TheresinwaswashedwithIPLysis/WashBuffer,1XConditioningBufferandelutedwithElutionBuffer.Foranalysis,4-20%Tris-glycinegelswereloadedwith20%oftheelutedsample,5%ofthecelllysateload(Lysate)and10%oftheantibodyload(IgG)andstainedwithThermoScientificImperialProteinStain(Product#24615).Fortheresincon-trols,theimmunoprecipitationwasperformedwithoutaddingtheantibody.
Co-immunoprecipitation of interacting proteins using the Thermo Scientific Pierce Co-IP Kit. EpidermalGrowthFactor(EGF)wasco-immunoprecipitatedfrom2,000μgofHeLalysatewith40and20μganti-EpidermalGrowthFactorReceptor(EGFR)mousemonoclonalIgG1+IgG2a(ThermoScientific)usingthePierceCo-IPKit.Co-immunoprecipitationswereperformedaccordingtoproductinstructions.TwoidenticalWesternblotswereprobedwithanti-EGFRsheeppolyclonalIgG(Millipore)oranti-EGFrabbitpolyclonalIgG(SantaCruz).ThePierceCo-IPKitco-immunoprecipitatedEGFwiththeEGF-R.
The Classic IP Kit:Product#26146• High binding-capacity recombinant Protein
A/G Plus Agarose Resin results in higher antigen yields
• Recombinant Protein A/G Plus Resin offers compatibility with a wider range of mamma-lian IgG species for IP reactions (e.g., mouse, rabbit, human and goat IgG subclasses)
The Crosslink IP Kit:Product#26147• Able to purify target protein without con-
tamination by the antibody in the eluate
• Improved crosslinking protocol optimized for maximum antibody functionality
• Bound immunoglobulins oriented for optimal antigen-binding sites are more accessible
• High-capacity Protein A/G Plus Resin allows for better purifi-cation and immobilization of antibodies
• Offers compatibility with a wider range of mammalian IgG species
The Direct IP Kit: Product#26148• Immobilize any antibodies independent
of isotype or species
• Improved antibody coupling protocol
• Purify target protein without antibody contamination
• Activated resin for directly coupling antibodies to the support resin
• Eliminate antibody contamination in the eluate
The Co-IP Kit: Product#26149• Minimal antibody requirements for
co-IP reactions
• Shorter antibody coupling protocol (<2 hours)
• Compatible with any antibody species and subclass
• Requires a purified antibody in a solution free of amines and stabilizing proteins
• Optimized protocols and buffers for efficient co-IP and antigen elution
• Allows for selective purification of target protein
• Includes spin columns and collection tubes that shorten the protocol by minimizing handling and mixing
• Compatible with specialized downstream applications, e.g., mass spectrometry
• Allows for scale up
Bead
Bead
Bead
Bead
Protein A/G PlusAntibodies Alkylamine linkage DSS Crosslinking
GFP (27 kDa)
IgG Heavy Chain(55kDa)
Thermo ScientificPierce IP Kits
Direct
Classic
Lysa
teIgG MW
Direct
Classic
Crosslin
k
Crosslin
k
IgG Light Chain(25kDa)
Resin Controls
EGF-R (170kDa)
µg anti-EGFR
EGF (6kDa)
40 20
IP/Co-IP
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 41
Thermo Scientific Pierce Classic IP KitA convenient kit for a new spin on traditional immunoprecipitation.
ThePierceClassicIPKitprovidesallthenecessaryreagents,spincupsandcollectiontubestoperformsuccessfulimmunopre-cipitation(IP)experimentswithease.Thekituseshigh-capacityProteinA/GagaroseaffinityresinforefficientbindingofmostspeciesandsubclassesofIPantibodies.TheincludedIgGelu-tionbufferprovidesmilderandlessdenaturingrecoveryofantibody:antigencomplexesthanthetraditionalmethodofboilinginreducingsamplebufferforSDS-PAGE,facilitatingagreatervarietyofmethodsforsubsequentanalysis.Themicrocentrifugespincolumnformathelpstoensureeffectivewashingandsep-arationofsamplesfromthebeadedagaroseaffinityresin.
LiketraditionalIPmethods,thePierceClassicIPKitprocedureinvolvesformationofantibody:antigencomplexesinasamplesolutionandthencaptureofthatcomplextoanIgG-bindingproteinthatiscovalentlyboundtobeadedagaroseresin(ProteinA/GAgarose).Afterwashingtoremovenonbound(presumablyundesired)componentsofthesample,theantigenandantibodyarerecoveredfromthebeadedresinwithelutionbuffersuppliedinthekit.Theentireprocedureisperformedinamicrocentrifugespincolumn,allowingsolutionstobefullyseparatedfromtheagaroseresinuponbriefcentrifugation.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
26146 Pierce Classic IP Kit Sufficient reagents to perform 50 reactions. Includes:PierceProteinA/GPlusAgarose
IPLysis/WashBuffer100XConditioningBuffer20XTris-BufferedSalineElutionBuffer5XLaneMarkerSampleBuffer,Non-reducingPierceSpinColumns–ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubesPierceControlAgaroseResin
Kit0.55mL2x50mL5mL25mL50mL5mL
50each2mL,100each1.5mL,50each2mL
$259
Thermo Scientific Pierce Crosslink IP KitPurify target protein complexes without antibody interference!
TheCrosslinkIPKitextendsthefunctionalityoftraditionalimmuno-precipitation(IP)methodsbyaddingcrosslinkingtechnologyandmicrocentrifugespincolumnsamplehandlingtotheprocedure.Theprimarybenefitsresultingfromthesefeaturesaretheabilitytopurifytargetproteinwithoutcontaminationbytheantibodyandtheabilitytomoreeffectivelywashandseparatesamplesfromthebeadedagaroseresin.
ThePierceCrosslinkIPKitmethodinvolvescapturingtheIPanti-bodytoProteinA/GAgaroseresinandcovalentlyimmobilizingittothesupportbycrosslinkingwithdisuccinmidylsuberate(DSS).Theantibodyresinisthenincubatedwiththesamplethatcontainstheproteinantigenofinterest,allowingtheantibody:antigencom-plextoform.Afterwashingtoremovenonbound(presumablyundesired)componentsofthesample,theantigenisrecoveredbydissociationfromtheantibodywithelutionbuffersuppliedinthekit.Theentireprocedureisperformedinamicrocentrifugespincup,allowingsolutionstobefullyseparatedfromtheagaroseresinuponbriefcentrifugation.Onlyantigeniselutedbytheprocedure,enablingittobeidentifiedandfurtheranalyzedwithoutinterfer-encefromantibodyfragments.Furthermore,theantibodyresinoftencanbereusedforadditionalroundsofimmunoprecipitation.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
26147 Pierce Crosslink IP Kit Sufficient reagents to perform 50 reactions. Includes:PierceProteinA/GPlusAgarose
20XCouplingBufferDSSCrosslinker,No-Weigh™FormatIPLysis/WashBuffer100XConditioningBuffer20XTris-BufferedSalineElutionBufferLaneMarkerSampleBuffer,Non-reducing,(5X)PierceSpinColumns-ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubesPierceControlAgaroseResin
Kit0.55mL25mL8x2mg2x50mL5mL25mL50mL5mL
50each2mL,100each1.5mL,50each2mL
$305
42 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Thermo Scientific Pierce Direct IP KitImmunoprecipitate using any antibody species or subclass! Eliminate antibody band contamination of IP products.
ThePierceDirectIPKitrepresentsasignificantadvancementinimmunoprecipitation(IP)technologybyreplacingtheuseofimmobilizedProteinAorProteinGwithamethodfordirectcovalentattachmentofantibodiestothebeadedagaroseresin.
Theprimarybenefitsresultingfromthismethodaretheopportunitytouseanyspeciesorsubclassofpurifiedantibody(notjusttypesthatbindtoProteinAorG)andtheabilitytopurifytargetproteinwithoutcontaminationbytheantibody.Themethodalsomakesitpossibletoimmunoprecipitateantigensfromserumsampleswithoutco-purifyingnon-targetimmunoglobulins.Finally,thekitusesmicrocentrifugespincupstoeffectivelywashandseparatesamplesfromthebeadedagaroseresin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
26148 Pierce Direct IP Kit Sufficient reagents to perform 50 reactions. Includes:AminoLinkPlusCouplingResin
20XCouplingBufferQuenchingBufferWashSolution5MSodiumCyanoborohydrideSolutionIPLysis/WashBuffer100XConditioningBuffer20XTris-BufferedSalineElutionBuffer5XLaneMarkerSampleBufferPierceSpinColumns–ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubesPierceControlAgaroseResin
Kit2mL25mL50mL50mL0.5mL2x50mL5mL25mL50mL5mL50each2mL,100each1.5mL,50each2mL
$259
Thermo Scientific Pierce Co-Immunoprecipitation KitPerform co-immunoprecipitation experiments without antibody interference.
ThePierceCo-Immunoprecipitation(Co-IP)Kitenablesisolationofnativeproteincomplexesfromalysateorothercomplexmixturebydirectlyimmobilizingpurifiedantibodiesontoanagarosesupport.
Co-IPisacommonapproachtostudyprotein:proteininterac-tionsthatusesanantibodytoimmunoprecipitatetheantigen(baitprotein)andco-immunoprecipitateanyinteractingproteins(preyproteins).Traditionalco-IPmethodsthatuseProteinAorGresultinco-elutionoftheantibodyheavyandlightchainsthatmayco-migratewithrelevantbands,maskingimportantresults.ThePierceCo-IPKitresolvesthisissuebycovalentlycouplinganti-bodiesontoanamine-reactiveresin.Thekitincludesoptimizedbuffersforproteinbindingandrecovery,reagentstoperformcon-trolexperimentsandefficientspincolumnsandcollectiontubes,whichshortentheprotocolandminimizehandlingandmixing.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
26149 Pierce Co-Immunoprecipitation Kit* Sufficient reagents to perform 50 reactions. Includes:AminoLinkPlusCouplingResin
20XCouplingBuffer5MSodiumCyanoborohydrideSolutionQuenchingBufferWashSolutionIPLysis/WashBuffer100XConditioningBufferElutionBuffer5XLaneMarkerSampleBuffer,Non-reducingPierceControlAgaroseResin20XModifiedDulbecco’sPBSBufferPierceSpinColumns–ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubes
Kit2mL25mL0.5mL50mL60mL2x50mL5mL50mL5mL2mL25mL50each100each50each
$308
* The Thermo Scientific Pierce Co-IP Kit (Product # 23600) and the Pierce Mammalian Co-IP Kit (Product # 23605) were discontinued on December 31, 2009. The next-generation Pierce Co-IP Kit (Product # 26149) will replace these old kits. The new co-IP kit is optimized for using smaller amounts of sample and offers a common lysis/wash buffer eliminating the need for a separate lysis reagent (Thermo Scientific M-PER Mammalian Protein Extraction Reagent, Product # 78503) as was offered with the Mammalian Co-IP Kit.
IP/Co-IP
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 43
Thermo Scientific Pierce Coated Plate Immunoprecipitation KitsPre-coated 96-well plates are easier to use and faster than traditional microcentrifuge tube methods.
ThermoScientificPierceCoatedPlateIPKitsenablerapidimmunoprecipitationofmultiplesampleswithouttheusualtediumofpipetting,centrifugingandseparatingbeadedaffinityresininindividualmicrocentrifugetubes.Immunoprecipitationisaccomplishedusingcoated96-wellmicroplatesratherthanbeadedagaroseresin.Theplateformatallowsfastprocessingofmultiplesamples.SelectfromProteinA/G-,ProteinG-orstreptavidin-coatedplates.
Highlights:•Ready-to-use,high-qualitycoatedplatesprovidehighcapacity andconsistency•Plateformatbestsuitedforsimultaneouslyprocessingmultiple samplesandtheircontrolconditions•Faster,easierandmorethoroughwashingthanwithtraditional tube/resinIPmethods•UsesfamiliarandconvenientELISAtools(multichannelpipettors andplatewashing);notediousseparationofsupernatantfrom pelletedresinbeads,andnotubestoopen,closeandcentrifuge• Coatedplatesare96-wellstripplates,convenientforexperi-
mentsrequiringonlyapartialplate•Easy-to-followinstructions,includingdetailedexplanationof appropriatecontrols•Threekitsavailable,suitableformostcommonantibodytypes (mouse,rabbit,humanandgoatIgGsubclasses)orany biotinylatedantibodyor“bait”protein
3 4 521
Thermo Scientific Pierce Protein A/G Coated Plate IP Kit (Product # 45350) immuno precipitation of CD71 (transferring receptor) from human serum using and a goat anti-CD71 polyclonal antibody.Elutedproductsfortheexperimentalandcontrolsamplesweremixedwithnonreducingsampleloadingbuffer,separatedbySDS-PAGE,transferredtonitrocellulosemembraneanddetectedbyWesternblottingwiththeIPantibody,Goat-anti-mouse-HRPconjugatedsecondaryantibody(Product#31432)andThermoScientificSuperSignalWestDuraChemiluminescentSubstrate(Product#34076).Lane 1:Experiment(immunoprecipitationproduct)Lane 2:Antibody-onlycontrol(nosample)Lane 3:Humanserumsamplecontrol(noantibody)Lane 4:Platecontrol(noantibodyorhumansample)Lane 5:Puretargetprotein(CD71)forsizereference
Choosing between Protein A/G and Streptavidin Coated Plate KitsStreptavidinisaproteinthatbindsspecificallyandstronglytobiotin;therefore,theStreptavidinCoatedPlateIPKit(Product#45360)isappropriateforimmunoprecipitationwhenusingabiotin-labeled(biotinylated)antibody.Thiskitcanbeusedtoaffinity-purifyabindingpartnertoanyantibodyspeciesorsubclassoranyotherproteinormoleculethatisbiotinylated.Becausethestreptavidin-biotinaffinityinteractionissostrong,theelutionstepgenerallywilldissociateonlytheantigen(bindingpartner),notthebiotinylatedantibodyor“bait”protein.
ProteinAandProteinGaredifferentproteinsthatbindtoimmuno-globulins(primarilyIgG).Typically,ProteinAispreferredforusewithrabbitpolyclonalantibodies,whileProteinGispreferredforusewithmouseantibodies(especiallymonoclonalsoftheIgG1subclass).ProteinA/GisarecombinantofProteinAandProteinGthathastheadditivebindingpropertiesofbothproteins.
ReferenceDesai,S.andHermanson,G.(1997).Previews1(3),2-7.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
45350 Pierce Protein A/G Coated Plate IP KitAntibody binding capacity/well: 2.5µg. Sufficient capacity for downstream analysis of IP or co-IP proteins in-gel or by Western blot.Includes:ProteinA/GCoated12x8-wellstripplates Phosphatebufferedsaline Surfact-Amps®X-100(10%TritonX-100) Elutionbuffer Neutralizationbuffer Uncoated96-wellstripplates(white), (forsamplecollectionandneutralization) Platesealers
Kit
2plates2packs6x10mL50mL7mL2ea.
18sheets
$295
45360 Pierce Streptavidin Coated Plate IP KitAntibody binding capacity/well: 5µg. Sufficient capacity for downstream analysis of IP or co-IP proteins in-gel or by Western blot.Includes: HighBindingCapacityStreptavidin CoatedPlates Biotinblockingbuffer Phosphatebufferedsaline Surfact-AmpsX-100(10%TritonX-100) Elutionbuffer Neutralizationbuffer Uncoated96-wellstripplates(white), (forsamplecollectionandneutralization) Platesealers
Kit
2plates30mL2packs6x10mL50mL7mL2ea.
18sheets
$298
44 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Thermo Scientific Pierce HA- or c-Myc Tag IP/Co-Immunoprecipitation KitsNeed to perform IP or co-IP reactions with your HA- or c-Myc-tagged protein? Open box ... Read instructions ... Start performing an IP or co-IP. High-specificity immobilized antibodies make it easy.
Notagsaremorepopularformammaliansystemproteinexpres-sionthanHAorc-Myc.Althoughthesetagsareextremelypopular,akitthatallowsyoutoconvenientlyperformimmunoprecipitation(IP)orco-immunoprecipitation(co-IP)reactionsusingthesetagshasnotbeenavailable.ThePierceIP/Co-IPKitsincludeallneces-saryreagents,buffersandhardwarethatallowefficientpurifica-tionofataggedtargetprotein(i.e.,IP)orconfirmationofpotentialinteractionsindicatedbyyeasttwo-hybridresults(i.e.,co-IP).
Thesefourkits,whicharespecificallyforHA-andc-Myc-taggedproteins,allowyoutoeasilyperformanIPorco-IPexperimentwithminimaloptimization.High-affinity,high-specificityantibod-iesimmobilizedontoanagarosematrixareattheheartofthesenewkits.Inaddition,thekitscontainafullcomplementofbuffers,eluents,apositivecontrolandnecessaryhardwaretoefficientlyperformtheintendedapplication.
Highlights:IP and co-IP for HA- or c-Myc-tagged proteins directly out of the box.• DemonstratedutilityintheIPandco-IPapplicationbenefitsthe
noviceandexpert.Ourkitsincludeallessentialcomponentstoperformtheassays.There’snoneedtoformulateorvalidaterawmaterials.
Immobilized high-affinity antibodies with excellent specificity for the HA or c-Myc tag.• Theimmobilizedanti-HAandanti-c-Mycmonoclonalsprecipitate
theappropriatelytaggedproteinspecificallyandinhighyield,resultingincleanWesternblotdetection.
• Excellentresultswithaslittleas2.5μgofanti-HAantibodyand1μgofanti-c-MycantibodyintheIPmodewiththerespectivepositivecontrollysate.
• Limitspossibilityofnonspecificbindingtootherproteinsinthelysate.
• Eliminatescontaminationfromantibodyorantibodyfragmentsafterelutionoftheprecipitatedproteinortheco-IPcomplex.Thisbenefitisespeciallyimportantwheninterpretingproteininteractionresults.
Simple, flexible and easy-to-follow protocols.• Completekitformatoffersoptimumconvenienceinboththe
IPandco-IPmodes.• EliminateProteinAorProteinG,reducingnonspecificbinding
andshorteningtheIPprocedure.• Systemdemonstratesexcellentflexibilitywithrespecttothe
amountofantibodyoramountoflysateused,enablingisolationoflow-expressionHA-/c-Myc-taggedtargets.
• TheuseofSpinColumnsacceleratetheIP/co-IPprocess.Spin Columns.•SpinColumnsareveryconvenientforsmallsamplehandling.•Allowmoreefficientwashing.•Eliminateresinlosses
HA- or c-Myc Tag IP/Co-IP Kit Descriptions Eachkitlistedatrightconsistsoftwocomponents:anApplicationSetandaPositiveControlLysate.TheApplicationSetcontainstheimmobilizedsupportappropriateforthekitandalloftherequiredbuffers,eluentsandhardware.ThesecondcomponentisabacteriallysatecontaininganoverexpressedGSTwitheitherHAorc-MycastheC-terminaltag.ThemammalianversionofeachkitcontainsThermoScientificM-PERProteinExtractionReagentforusewithmammaliancell-basedIPorco-IPapplications.TheApplicationSetsandPositiveControlLysatescanalsobeorderedseparately.
1. Lyse cells expressing HA- or c-Myc-tagged protein.
2. Combine cell lysate and Immobilized Anti-HA or Anti-c-Myc.
3. Incubate at 4˚C for 1 hour to overnight with end-over-end mixing.
4. Pulse centrifuge for 10 seconds.
5. Wash three times with TBS-T. Pulse centrifuge for 10 seconds after each wash.
6. Elute HA- or c-Myc-tagged protein using Elution Buffer or Non-Reducing Sample Buffer.
CollectionTube
Bottom PlugRemoved
Thermo Scientific Pierce HA- or c-Myc IP/Co-IP Kit Protocol summary.
GST-c-MycGST-HA
IP
MW
Mar
ker
HA
Lys
ate
Elut
ion
#1
Elut
ion
#2
c-M
yc L
ysat
e
Elut
ion
#1
Elut
ion
#2
IP
Effectiveness of elution options in the IP of GST-HA and GST-c-Myc from bacterial lysates.IPresultsachievedwiththeThermoScientificPierceHAandc-MycIP/Co-IPKitsusingtheappropriatepositivecontrollysateprovidedandsuggestedelutionoptionsforGST-HAandGST-cMyc,respectively.Theelutioncomponentsaresuppliedwitheachkit.Elutionperformedwith#1.ElutionBufferor#2.2Xnonreducingsamplebuffer.
IP/Co-IP
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 45
Ordering Information Product #
Description
Pkg. Size
U.S. Price
23610 HA-Tag IP/Co-IP Kit Sufficient material to conduct 25 IP/co-IP assays using proteins expressed with an HA tag. Kit is supplied complete with an HA-tagged positive control lysate.Includes:ImmobilizedAnti-HA(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps M-PERMammalianProteinExtraction Reagent HA-TaggedPositiveControl
Kit
150μL1pack50mL5mL
25mL
500μL
$399
23615 Mammalian HA-Tag IP/Co-IP KitSufficient material to conduct 25 IP/co-IP assays using proteins expressed with an HA tag. Kit is supplied complete with a mammalian cell lysis buffer and an HA-tagged positive control lysate.Includes:ImmobilizedAnti-HA(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps M-PERMammalianProteinExtraction Reagent HA-TaggedPositiveControl
Kit
150μL1pack50mL5mL
25mL
500μL
$423
23620 c-Myc-Tag IP/Co-IP KitSufficient material to conduct 25 IP/co-IP assays using proteins expressed with a c-Myc tag. Kit is supplied complete with a c-Myc-tagged positive control lysate.Includes:ImmobilizedAnti-c-Myc(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps c-Myc-TaggedPositiveControl
Kit
250μL1pack50mL5mL
500μL
$399
23625 Mammalian c-Myc Tag IP/Co-IP KitSufficient material to conduct 25 IP/co-IP assays using proteins expressed with a c-Myc tag. Kit is supplied complete with a mammalian cell lysis buffer and a c-Myc-tagged positive control lysate.Includes:ImmobilizedAnti-c-Myc(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps M-PERMammalianProteinExtraction Reagent c-Myc-TaggedPositiveControl
Kit
250μL1pack50mL5mL
25mL
500μL
$423
Protein Interactions Technical Handbook
Our72-pageProteinInteractionTechnicalHandbookprovidesprotocolsandtechnicalandproductinformationtohelpmaximizeresultsforProteinInteractionstudies.Thehandbookprovidesbackground,helpfulhintsandtroubleshootingadviceforimmunoprecipitationandco-immunoprecipitationassays,pull-downassays,Far-Westernblottingandcrosslinking.Thehandbookalsofeaturesanexpandedsectiononmethodtostudyprotein:nucleicacid
interactions,includingChIP,EMSAandRNAEMSA.Thehand-bookisanessentialresourceforanylaboratorystudyingProteinInteractions.(#1601618)
46 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Protein Enrichment
Protein Enrichment
Protein enrichment encompasses numerous techniques to isolate subclasses of cellular proteins based on their unique biochemical activity, post-translational modification (PTM) or spatial localization in a cell. Protein enrichment is essential for studying low abundant proteins and for reducing the complexity of samples for proteomic analysis. Enrichmentofspecificproteinsorproteincomplexescanmosteasilybeaccomplishedusingimmunoaffinitytechniquessuchasimmunoprecipitationandco-immunoprecipitation.Althoughtheseantibody-basedtech-niquesarewidelyused,elutionofimmunoprecipitatedproteinscansometimesresultinlowproteinrecoveryorantibodycontami-nationinsamples.
Globalproteinenrichmentstrategiesinvolvetheselectiveisola-tionofdistinctproteinsubclasseswhichshareacommonpost-translationalmodificationorcellularlocalization.Post-translationalmodificationssuchasphosphorylationandglycosylationcanbeenrichedusingaffinityligandssuchasion-metalaffinitychroma-tography(IMAC)orimmobilizedlectins,respectively.Inaddition,PTM-specificantibodieshavebeenbeused.OthertechniquesusemetabolicorenzymaticincorporationofmodifiedaminoacidsorPTMstointroduceuniqueproteinchemistrywhichcanbeusedforenrichment.Finally,proteinscanalsobeenrichedusingvariousenzymeclassspecificcompoundsorcell-impermeablelabelingreagentswhichselectivelylabelcellsurfaceproteins.
Phosphoprotein Enrichment Kits
Process cell and tissue samples in less time and with greater purity.
Phosphorylationisoneofthemostfrequentlyoccurringpost-translationalmodificationsinproteins.Itisestimatedthatasmanyas30%ofallcellularproteinsaretransientlyphosphorylatedonserine,threonineandtyrosineresidues.
Reversibleproteinphosphorylationregulatesnearlyallintra-cellularbiologicalevents,includingsignaltransduction,protein-proteininteractions,proteinstability,proteinlocalization,apoptosisandcell-cyclecontrol.Deregulationofproteinphosphorylationisahallmarkofnumeroushumandiseases,includingcancerandmetabolicandimmunedisorders.
Detectingchangesinproteinphosphorylationcanbeadifficulttaskbecauseofthetransientlabilestateofthephosphategroup.Furthermore,lowphosphoproteinabundanceandpoorlydevelopedphospho-specificantibodiescontributetodifficultiesinphosphoproteindetection.Recentadvancesinmassspectrometrytechnologyincombinationwithphosphoproteinenrichmentusingimmobilizedmetalaffinitychromatography(IMAC)haveresultedingreaterresolutionofthephosphoproteome.
ThenewThermoScientificPiercePhosphoproteinEnrichmentKitefficientlyenrichesphosphorylatedproteinsderivedfrommammaliancellsandtissues.Theproprietarymetalandbuffercompositionproducessuperioryieldswithnegligiblenonspecificbinding.
Highlights:• Specific–lowcontaminationfromnonspecificproteins• Fast–easy-to-usespinformatenrichesofphosphorylated
proteinsinlessthan2hours• Superior yield–highyieldfromcomplexbiologicalsamples,cell
culturelysateandmousetissueextract• Convenient format–completekitincludespre-dispensedspin
columns,buffers,reagentsandThermoScientificPierceProteinConcentrators
• Compatible–workswithdownstreamapplications,includingmassspectrometry,Westernblottingand2D-PAGE
Phospho-specificantibodiesrecognizingkeyregulatoryproteinsinvolvedingrowthfactorsignalingwereusedtomonitorbindingspecificityofourPhosphoproteinEnrichmentKit(Figure1).SpecificityofthekitisfurtherdemonstratedbytheabsenceofCytochromeC(pI9.6)andp15Ink4b(pI5.5),twoproteinsnotpredictedtobephosphorylated,intheelutionfractionandtheiremergenceintheflow-throughandwashfractions(Figure1).Furthermore,dephosphorylationofHeLacellextractin vitroresultedindiminishedbindingofPTEN,MAPKandGSK3βtothePiercePhosphoproteinEnrichmentColumnasevidencedbytheirabsenceintheelutionfraction.Conversely,allthreeproteinswerepresentintheelutionfractionfromnon-treatedHeLaextract(Figure2).OurPhosphoproteinEnrichmentKitprovidedsuperiorandefficientphosphoproteinenrichmentyieldswhencomparedtocompetitors’products(Table1).Italsoeffectivelyenrichedphos-phoproteinsfromhomogenizedmouselivertissue(Figure3).
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 47
Table 1. The Thermo Scientific Pierce Phosphoprotein Enrichment Kit provides higher phosphoprotein yields in less time than competitors’ kits.
Kit Yield (%) Enrichment Time (Hours)
ThermoScientificPiercePhosphoproteinEnrichmentKit
15 1.5
SupplierQKit 4.4 4.5
SupplierIKit 2.6* 3.5
SupplierCKit 8 3
SupplierEKit Toodilutetodetermine 5
*Based on maximum 1mg load per manufacturer’s protocol.
Phospho-MAPK T202/Y204
Phospho-PTEN S380
Phospho-GSK3β S9
Phospho-MAPK T202/Y204
Phospho-Akt S473
Phospho-Src Y527
Cytochrome C
p15Ink4b
NIH
3T3
+ P
DG
F
H
eLa
+ EG
F
FT W E L
Figure 1. Highly pure phosphoprotein enrichment from complex biological samples. Serum-starvedHeLaandNIH3T3cellswerestimulatedwithEGFandPDGF,respectively.Celllysate(2mg)wasusedforenrichment.Concentratedflow-through,washandelutionfractionswereresolvedbySDS-PAGE.Gellaneswerenormalizedbyproteinconcentration(10μg/lane).Westernblotanalysiswasperformedusingantibodiesthatdetectsite-specificphosphory-lationevents.CytochromeC(pI9.6)andp15Ink4b(pI5.5)servedasnegativecontrolsfornonspecificbindingofnon-phosphorylatedproteins.FT =flow-throughfraction,W =pooledwashfractions,E =pooledelutionfractionsandL =non-enrichedtotalcelllysate.
Phospho-PTEN S380
Phospho-MAPK T202/Y204
Phospho-GSK3β S9
Total Cell Lysate Elution25 µg 10 µg 10 µg
λ Pase - + - + - +
Figure 2. Highly specific phosphoprotein purification from lambda phosphatase-treated cells.Non-treatedandlambdadephosphorylatedHeLacellextract(2mg)wasloadedontoseparateThermoScientificPiercePhosphoproteinEnrichmentColumns.ConcentratedelutionfractionswereresolvedbySDS-PAGE.Gellaneswerenormalizedbyproteinconcentration(10μg/lane).Todetermineenrichment,10μgand25μgofnon-treatedorlambdaphosphatase-treatedtotalcellextract(non-enriched)wasloadedontoeachgel.Westernblotanalysiswasperformedusingphospho-specificantibodiesrecognizingkeyproteinsintheRas-MAPKandPI3K-Aktsignalingcascades.
L10 FT W E L25
Phospho-PTEN S380
Phospho-Rb S795
Cytochrome C
Figure 3. Efficient enrichment of phosphoproteins from mouse liver extract. Homogenizedmouseliverextract(~2mg)wasloadedontoaThermoScientificPiercePhosphoproteinEnrichmentColumn.Concentratedflow-through,washandelutionfractionswereresolvedbySDS-PAGE.Gellaneswerenormalizedbyproteinconcentration(10μg/lane).Westernblotanalysiswasperformedusingantibodiesthatdetectsite-specificphosphorylationevents.CytochromeC(pI9.6)servedasanegativecontrolfornonspecificbinding.L10 =non-enrichedtotalcellextract(10μg),FT =flow-throughfraction,W =washfraction,E =elutionfractionandL25 =non-enrichedtotalcellextract(25μg).
Ordering Information Product #
Description
Pkg. Size
U.S. Price
90003
Pierce Phosphoprotein Enrichment KitIncludes:PhosphoproteinEnrichmentColumn
ResinBed(1mL)Lysis/Binding/WashBufferElutionBufferCHAPSWhiteColumnCapsPierceProteinConcentrator7mL/9KMWCO
Kit10ea.325mL60mL1g10caps10Devices
$464
48 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Phosphoprotein Pull-Down with SH2 Domains
SH2 Domain Phosphotyrosine Capture Kits
SH2domainsprovideanimprovedapproachtoselectivelymonitorreceptortyrosinekinasesignaling,aswellasthebindingofdown-streameffectorproteins.TheinteractionsofSH2domain-contain-ingproteinsrepresentacriticalinterfacebetweenextracellularstimulationofamembranereceptorandtransmissionofthatsignaltointracellularproteins.
OurkitsincludeoptimallevelsofpurifiedGST-fusedSH2domainsofvarioussignalingproteinsintegraltocellbiology.Eachvalidatedkitincludesoptimizedbuffersandcolumnstoperformproteinpull-downs.UsingSH2domainseliminatesthebackgroundassociatedwithlow-specificityantibodiesandenablesanalysisofreceptortargetstowhichantibodiesarenotavailable.Usingquality-testedpurifiedGST-SH2domainsalsoensuresuniformresultswithoutvariability.
SH2 domains specifically bind phospho-tyrosine residues. EachSH2domainisspecificforitsnaturaltarget(Figure4).In vivo,thephosphataseShp2isrecruitedtotyrosine1009oftheplatelet-derivedgrowthfactorreceptor(PDGFR)onlywhenitisphosphorylated.Also,PLCgisrecruitedtotyrosine1021onPDGFRinresponsetogrowthfactorsignaling.Inquiescentcellsbothtyrosine1009and1021arenotphosphorylatedand,therefore,bothdomainsareunabletobind.
PDGF
Antibody
GST-Shp2 GST-PLCγ
L PD L PD
- +
L PD L PD
- +
pPDGFR Y1009 pPDGFR Y1021
Figure 4. Site-specific interaction of GST-Shp2 and GST-PLCg SH2 on the PDGF-receptor.NIH3T3cellswererenderedquiescentbyserumwithdrawalfor48hoursfollowedbystimulationwithPDGF(50ng/mL,CellSignalingTechnology[CST])for15minutesornostimulation.Eachcelllysate(500μg)wasincubatedovernightat4°Cwitheither100μgGST-PLCg1orGST-Shp2.ProteincomplexeswerecapturedonimmobilizedglutathionebeadsandresolvedbySDS-PAGE.Westernblotanalysiswasperformedusingphospho-specificantibodiesthatdetectknownprotein-proteininteractionsequences(phospho-PDGFRY1009andphospho-PDGFRY1021,CST).Lanes:L = 25μgoftotalcelllysateandPD =SH2domainpull-down.
pY
pY
pY
pY
pY
pY SH2
SH2
Receptor in cell membrane
Extracellular signal
Tyrosine phosphorylation
SH2 domains recognize pY
Other proteins recruited
Translation, Transcription, Cell Proliferation, Differentiation, Apoptosis
Cellular pathway activation
SH2 domains provided a more efficient method of capturing site-specific phospho-tyrosine events as compared to antibody based co-immunoprecipitation. Co-immunoprecipitationexperimentsaredependentonthequalityandspecificityoftheantibodyused.Antibodiesagainstphosphoproteinsarenotoriouslydifficulttoworkwith,yieldinghighbackgroundandlowtargetproteinrecovery.SH2domainsprovidebettercaptureefficiencyascomparedtomanypan-andsite-specificphospho-antibodiesbecauseanSH2domainismoreselectivebynature.Becauseofthisimprovedspecificity,SH2domainscanbeusedtopull-downphosphotyrosinecontainingproteinswithimprovedresultsovertraditionalantibody-basedmethods.InFigure5,PDGF-stimulatedNIH3T3cellswerelysedandincubatedwitheitheraspecificSH2domainorapan-antibodycorrespondingtotheSH2domaincontainingprotein.BindingofeachspecificSH2domaintothePDGFreceptorwasconfirmedbyWesternblotusingapan-PDGFRantibody.InallcasesweobservedahighercaptureefficiencyusingtheSH2domainapproachascomparedtothetraditionalantibodyco-immunopre-cipitation.Inmanycases,wewerealsoabletodetectdifferentialbindingoftheSH2domaininthepresenceofPDGFstimulationascomparedtotheunstimulatedstate.Thesedifferenceswerenotapparentusingtheco-immunoprecipitationapproach.WefurthertestedtheinteractionspecificityoffourSH2domainsusingphospho-antibodieswhichdetectsite-specifictyrosinephosphorylationeventswhichmediateeachSH2domaininteraction.Wewerelimitedtoonlyfourphospho-antibodiesagainstspecifictyrosinesitesonthePDGFreceptorbecauseofcommercialavailabilityand/orantibodyintegrity.Usingthesesite-specificantibodiesweconfirmedtheselectivityandspecificityofeachSH2domaintestedandtheseinteractionsweremuchstrongerascomparedtothetraditionalantibodyimmunoprecipi-tation.TakentogethertheseresultsclearlydemonstratethattheSH2domainpull-downapproachrepresentsarobustmethodtocaptureandmonitorsitespecifictyrosinephosphorylationeventswhicharecriticalforcellhealth.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 49
Figure 5. SH2 domains provide better enrichment of phosphorylated receptor tyrosine kinase (PDGFR) compared to antibody-based pull-down assays. Panel A. NIH3T3cellswererenderedquiescentbyserumstarvationfor24hourswithDMEMcontaining0.1%FCS.Followingstarvationcellswereeitherstimulated(100ng/mLPDGFfor20minutes)orleftunstimulated.CellswerethenlysedinNP40lysisbuffer(50mmTrispH7.5,150mmNaCl,1%NP40,5%glycerol)containingThermoScientificHaltProteaseandPhosphataseInhibitorCocktail(Product#78440)andproteinconcentra-tionsdeterminedbytheThermoScientificPierce660nmAssay.SH2andco-immunoprecipitationswereperformedasfollows:SH2domainpull-downsconsistedof100μgofSH2domain(Abl,Crk,Grb2,Lyn,PI3K,Plc-gamma,RasGap,Shc,Shp2,Src)addedto250μgofstimulatedorunstimulatedlysate.Co-immunoprecipitationswereperformedbyincubating10μLofpanantibody(Abl,Crk,Grb2,Lyn,PI3K,Plc-gamma,RasGap,Shc,Shp2,Src)with250μgofstimulatedornon-stimulatedlysate.Bindingwasperformedfor16hoursat4°Conarotatingplatform.Followingbinding100μLofa50%slurryofimmobilizedglutathionewasaddedtotheSH2domainsamplesand100μLofa50%slurryofProteinA/Gwasaddedtotheco-immunoprecipitationsamples.Sampleswere
incubatedfor1hourat4°Conarockingplatformfollowedbycentrifugationat1000xgfor1minutetocollectcomplexesboundtotheresin.Resinbedswerethenwashed3timeswith500μLofThermoScientificM-PERWashBuffer.Toeluteboundproteins25μLof5XDTTloadingdyewasaddedtoeachsampleandboiledfor5minutes.ProteincomplexeswerethenresolvedbySDS-PAGEona4-20%Tris-glycinegel.ProteinsweretransferredontoPVDFmembranefor16hoursat4°C,30V.Membraneswereblockedin7.5%BSAinTBSTandWesternblotsperformedusinga1:2000dilutionofmousemonoclonalanti-PDGFR-betaantibody(CST3175).DetectionwasperformedusingThermoScientificSuperSignalPicoChemilumescentDetectionSystem.Panel B. MembranescontainingtheGrb2,Plcg,PI3K,Src,andRasGappull-downswerestrippedwithThermoScientificRestorePlusBufferandreprobedwithphospho-antibodieswhichdetectsite-specifictyrosinephos-phorylationeventsonthePDGFreceptorwhichmediatetheSH2domaininteraction.TheseantibodiesincludepPDGFRY716,pPDGFRY751,pPDGFRY1009,pPDGFRY579whichcorrespondtoSH2domaindockingsitesforGrb2,PI3K,Plcg,andSrcrespectively.DetectionwasperformedusingSuperSignal®WestPicoChemilumescentDetectionSystem.
PDGF - + - + - + - + - + - + - + - + - + - +
PDGF - + - + - + - + - + - + - + - + - + - +
SH2 IP SH2 IP SH2 IP SH2 IP SH2 IP
SH2 IP SH2 IP SH2 IP SH2 IP SH2 IP
Abl Crk Grb2 Lyn PI3K
Plcγ RasGap Shc Shp2 Src
Panel A.
Panel B.
Western Blot: Pan PDGFR
Western Blot: Pan PDGFR
PDGFR
PDGFR
SH2: Grb2IP: Pan Grb2 WB: pPDGFRY716
SH2: PI3KIP: Pan PI3K WB: pPDGFRY751
SH2: PlcγIP: Pan PlcγWB: pPDGFRY1009
SH2: Src IP: Pan Src WB: pPDGFRY579
PDGF - + - + - + - + - + - + - + - +SH2 IP SH2 IP SH2 IP SH2 IP
pPDGFR
50 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Perform receptor phosphorylation time-course experiments. WithSH2domainpull-downs,itispossibletomonitortime-dependentbindingofSH2domainstotheirtarget.Forexample,PLCg,SrcandShcbindrapidlytothePDGFreceptorinresponsetoPDGFstimulation(Figure6).Innature,phosphorylationisrapidandtran-sient,asevidencedbydiminishedbindingat10-and20-minutepost-stimulation.
MinutesPost PDGF
Stimulation
GST-PLCγ
GST-Src
GST-Shc
0 1 5 10 20
Figure 6. Time-specific binding of various SH2 domains to the PDGF receptor. NIH3T3cellswererenderedquiescentbyserumwithdrawalfor48hours.Post-starvationcellswerestimulatedwithPDGF(50ng/mL,CST)fortheindicatedtimesoruntreated.SH2domainpull-downswereperformedwith100μgofeachSH2domainand250μgofeachcelllysate.ProteincomplexeswereresolvedbySDS-PAGEandanalyzedbyWesternblotusingapanantibodyrecognizingPDGFR(CST).
Resolve specific tyrosine phosphorylation events. SH2domainaffinityprovidesalevelofselectivitynotpreviouslyattainablewithgenericanti-phosphotyrosineantibodies.UsedifferentSH2domainstouncoverthemechanismofhowsignalsaretransducedortoscreenmultipleSH2domainsagainstaparticulartargetatonetime.Forexample,differentnaturallyoccurringSH2domainsefficientlybindtotheepidermalgrowthfactorreceptor(EGFR)inresponsetoEGFstimulation(Figure7).Therewerestronginteractionsofmanyofthedomainsinthepresenceofepidermalgrowthfactor(EGF)andlow-levelbindinginquiescent,serum-starvedA431cells.RasGapbindingisindependentofEGFstimulation,whereasGrb2bindingishighlyupregulated(Figure7).
Ordering Information Product #
Description
Pkg. Size
U.S. Price
87700 Grb2 SH2 Domain Phosphotyrosine Capture KitSufficient reagents for six pull-down reactions (100µg/pull-down).Includes:GST-Grb2SH2Domain(37kDa)
GST(negativecontrol,27kDa)ImmobilizedGlutathioneResin(50%resinslurry)SpinColumn(0.8mL)M-PERMammalianProteinExtractionReagentLaneMarkerReducingSampleBuffer(5X)
6pull-downs
600μgat1mg/mL200μgat1mg/mL400μL8each25mL200μL
$408
87701 Src SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87702 Abl SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87703 Crk SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87704 Fyn SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87705 Lck SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
L25
*
* * *
* **
EGF
EGF
+ – + – + – + – + – + –
EGF
* = Known Interactions
Antibody: Pan EGFR
** = GST Only: Negative Control
L25+ –
Shp2+ –
Grb2 Src PLCγ Hck Fyn
L25+ – + – + – + – + – + –
Gap Abl Crk GST Nck
Figure 7. High efficiency binding of different naturally occurring SH2 domains to EGFR in response to EGF stimulation. A431cellswererenderedquiescentbyserumwithdrawalfor48hoursfollowedbystimulationwithEGF(100ng/mL)for15minutesorleftuntreated.Eachcelllysate(500μg)wasincu-batedwith100μgofeachGST-SH2domainovernightat4˚C.ProteincomplexeswerecapturedonimmobilizedglutathionebeadsandresolvedbySDS-PAGE.WesternblotanalysiswasperformedusingapanantibodythatrecognizestheEGFreceptor.L25=25μgtotallysateload.
Identify binding interactions with MS. Pull-downsamplesisolatedwithSH2domainsarecompatiblewithMSanalysisforidentifyingproteininteractingpartners.
ImprovedspecificitywithSH2domainsandtheeliminationofantibodycontaminationtypicallyseenintraditionalimmunopre-cipitationexperimentsdeliverpureproteinforMSanalysis.TwodifferentSH2domainswereusedtopulldowninteractingproteinsfromNIH3T3celllysates,whichwerethenanalyzedbyMS.ManyoftheinteractingproteinsidentifiedarespecifictoeitherFgrorPLCg.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
87706 Nck SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87707 Shc SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87708 Ras-GAP SH2 Domain Phosphotyrosine Capture Kit
6pull-downs $408
87709 Shp2 SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87710 PLC SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87711 Lyn SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87712 Hck SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87713 FGR SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87714 Cbl SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87715 Syk SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
87716 PI3K SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408
Phosphoprotein Pull-Down with SH2 Domains
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 51
Use of Titanium Dioxide for Phosphopeptide Enrichment
TitaniumdioxideenrichmentofphosphopeptidesamplesisanessentialtoolforcomprehensiveMSanalysisofphosphopeptides.TheuniquephosphopeptidebindingpropertiesofTiO2resinresultsindifferentselectivityandpreferentialenrichmentofmultiplyphosphorylatedpeptidescomparedtoimmobilizedchelatedmetalions(IMAC).TiO2resintypicallyenrichesmorephosphopeptidesthanIMACresins,andTiO2preferentiallyenrichesmonophos-phorylatedpeptides.
TiO2resintypicallybindsphosphopeptideswithahigheraffinitythanIMACandthepreferentialenrichmentofsinglyphosphory-latedpeptideswithTiO2islikelyduetodifficultyinelutingmultiplyphosphorylatedpeptides.Toenrichamorecomprehensivesetofphosphopeptides,theflow-throughandwashesfromIMACresincanbeacidifiedandappliedtoTiO2resinsandtheresultingelu-atespooledforanalysis.Inaddition,thenumberofphosphopep-tidesrecoveredcanbesignificantlyincreasedbypre-fractionationofsampleswithstrongcationexchangechromatographytoreducesamplecomplexitypriortoenrichmentwithTiO2orIMACprocessing.
Thermo Scientific Pierce TiO2 Phosphopeptide Enrichment and Clean-up KitSelective enrichment and Clean-up of phosphopeptides in under 2 hours.
ThePierceTiO2PhosphopeptideEnrichmentandClean-upKitenablesfast,selectiveenrichmentofphosphorylatedpeptides.Thecompletekitincludes24titaniumdioxide(TiO2)spintipsandgraphitespincolumnswithbufferstofacilitatepreparationofenrichedanddesaltedphosphopeptidesforanalysisbymassspectrometry(MS).
Highlights:• Convenient–spin-columnformatofTiO2andgraphitecolumns
enableparallelprocessingandcleanupofmultiplesamplesinlessthan2hours
• High capacity–eachcolumnenrichesupto300μgofphosphopeptides
• Complementary–TiO2enrichesauniquesetofphosphopeptidesthatcomplementsthePierceFe-NTAIMACPhosphopeptideEnrichmentKit
•High selectivity–recoverphosphopeptideswith>90%selectivity
ThePierceTiO2PhosphopeptideEnrichmentandClean-upKitprotocolincludesstringentwashingconditionsthatincreasetheselectivityforphosphopeptidesto>95%.Becausesomeofthesaltsinthesestringentwashesmaystillbepresentintheelutedsamples,thePierceTiO2PhosphopeptideEnrichmentandClean-upKitincludesgraphitespincolumnstodesaltandconcentrateenrichedphosphopeptides,resultinginmoresuccessfulphos-phopeptideanalysisresults.Thiskitiscompatiblewithsamplesdigestedinsolutionorafterin-geldigestionwithtrypsinorotherMSgradeproteases.UsingthePierceTiO2PhosphopeptideEnrichmentandClean-upKitandthePierceFe-NTAIMACPhosphopeptideEnrichmentKitswillenablecomplementarysetsofphosphopeptidestoberemovedfromcomplexsamples.
Number of Phosphates per Pepti0de
Resin 1 2 3 4 5 6 Total
TiO2 492 103 8 4 0 1 608
Fe-NTA IMAC 234 34 216 3 1 0 488
Overlap 155 0 1 0 0 0 156
Selective enrichment of singly and multiply phosphorylated phosphopeptides with Thermo Scientific Pierce TiO2 and FE-NTA IMAC resins. Averagephos-phopeptideenrichmentresultsfromduplicateexperimentsshowingthenumberofphosphopeptidescontainingoneormorephosphateperpeptideenrichedusingeitherresin.PeptidespectrumsummaryresultswereexportedfromProteomeSoftwareScaffold3.0.
120
100
80
60
40
20
Total PhosphopeptidesUnique Phosphopeptides% Acidic Residues
0
TiO2 Fe-NTA
Phos
phop
eptid
es (%
)
961 240
998238
TiO2 selectively enriches more phosphopeptides than Fe-NTA IMAC.Thenum-bersrefertothetotalanduniquenumberofphosphopeptidesenrichedbyeachmethod.TheY-axisisthepercentageofphosphopeptidesinthenumberoftotalanduniquepeptides,orthepercentageofacidicresiduesinenricheduniquephosphopeptides.
References:LarsenM.R.,et al.(2005).Highlyselectiveenrichmentofphosphorylatedpeptidesfrompeptidemixturesusingtitaniumdioxidemicrocolumns.Mol Cell Proteomics. 4(7):873-86.Carrascal,M.,et al.(2008).PhosphorylationAnalysisofPrimaryHumanTLymphocytesUsingSequentialIMACandTitaniumOxideEnrichment.J. Proteome Res.7(12):5167-5176.Wilson-Grady,J.T., et al.(2008).PhosphoproteomeAnalysisofFissionYeast.J. Proteome Res. 7(3):1088-1097.Larsen,M.R.,et al.(2004).Improveddetectionofhydrophilicphosphopeptidesusinggraphitepowdermicrocolumnsandmassspectrometry:evidenceforinvivodoublyphos-phorylateddynaminIanddynaminIII.Mol. Cell Proteomics.3:456-465.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
88301 Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit Sufficient For: 24 phosphopeptide enrichment and cleanup reactions. KitContents:TiO2SpinTips,24tips
TrifluoroaceticAcid,1mL90%LacticAcid,2mLPyrrolidine,200μLCentrifugeColumnAdaptors,24adaptorsPierceGraphiteSpinColumns,24columns
24-RxnKit $375
Phosphopeptide Enrichment
52 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Phosphopeptide Enrichment
Thermo Scientific Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment KitTiO2 magnetic particles for high throughput phosphopeptide isolation.
ThePierceMagneticTitaniumDioxidePhosphopeptideEnrichmentKitisforisolatingphosphopeptidesfromcomplexbiologicalsam-plesusingtitaniumdioxide-coatedmagneticbeads.TheTiO2ligandselectivelybindspeptidescon-tainingphosphorylatedserine(Ser),tyrosine(Tyr)orthreonine(Thr),enablingphosphopeptideenrichmentfromprotease-digestedsamples.Theisolatedphos-phopeptidesarecompatibleforanalysisdownstreambymassspectrometry(Table2).
Thehigh-performance,ironoxide,superparamagneticparticlesarevalidatedandoptimizedforusewithhigh-throughputmagneticplatforms,suchastheThermoScientificKingFisher96andKingFisher®FlexInstruments.Thebeadsalsoenablepremiumperformanceforsimplebenchtopapplicationsusinganappropriatemagneticstand.
Highlights:• Complete MS-compatible Kits–includeready-to-usebinding,
washandelutionbuffersthatareoptimizedforphosphopeptideenrichmentanddownstreamanalysisbyMALDIandESImassspectrometry
• Optimized for HTS–procedurevalidatedforprocessing1to96samplesatatime;completeentireassayinabout15minutesusingaKingFisherFlexInstrument
• Stable affinity ligand–titaniumdioxideisspeciallycoatedasafilmonthemagneticparticles
• Selective–affinitysystemisselectiveforphosphorylatedSer,TyrandThr;exhibitsminimalnon-specificbindingtoacidicresidues
• Sensitive–affinityprovidesmorethan1000timesgreatersensi-tivitythantraditionalIMACtechnologies;enablesenrichmentandMS-measurementoflessthan100fmolofphosphoprotein
Table 2. Phosphopeptide enrichment improves MS-identification of phospho-proteins. Twomilligramsofatrypticdigestpreparedfromperiferalbloodmono-nuclearcells(lymphocytes)withandwithoutphosphopeptideenrichmentwereanalyzedbyMS.EnrichmentwasperformedwiththeThermoScientificPierceTitaniumDioxidePhosphopeptideEnrichmentKitusingtheThermoScientificKingFisher96Instrument.SampleswereanalyzedonaThermoScientificLTQOrbitrapMassSpectrometer.
Enriched Non-Enriched
Totalnumberofproteinsidentified 185 247
Totalnumberofphosphoproteinsidentified 160 1
Totalnumberofpeptidesidentified 2347 2457
Totalnumberofphosphopeptidesidentified 2009 7
Totalnumberofuniquephosphopeptidesidentified 177 1
Relativeenrichmentforphosphopeptides(%) 86 0.3
Ordering Information Product #
Description
Pkg. Size
U.S. Price
88811 Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment KitIncludes:TiO2MagneticBeads(20X)
BindingBufferWashingBufferElutionBufferThermo-Fast96RoboticPCRPlate(0.2mLwells)
Kit1mL100mL25mL3mL2plates
$427
88812 Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit, Trial SizeIncludes:TiO2MagneticBeads(20X)
BindingBufferWashingBufferElutionBufferThermo-Fast96RoboticPCRPlate(0.2mLwells)
Kit0.25mL100mL25mL3mL2plates
$240
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 53
Thermo Scientific Pierce Fe-NTA Phosphopeptide Enrichment KitNew Fe-NTA format optimized for capture and recovery of phosphopeptides.
ThenewPierceFe-NTAPhosphopeptideEnrichmentKitenablesfastandefficientenrichmentofphosphorylatedpeptides.Thesespincolumnsareeasytouseandrequirelessthan1hourtoprocessproteindigestsorstrongcation-exchangepeptidefractionsforanalysisbymassspectrometry(MS).
Highlights:•Convenientspinformatforparallelprocessingof
multiplesamples•High-bindingcapacityresinforenrichingupto150μg
ofphosphopeptidespercolumn•Excellentenrichmentandrecoveryofphosphopeptides
Proteinphosphorylationisessentialtobiologicalfunctions,includingcellsignaling,growth,differentiation,divisionandprogrammedcelldeath.Over500proteinkinasescatalyzephosphorylationofspecifictargets,primarilyonserine,threonine,andtyrosineresidues.
Massspectrometryisincreasinglybeingusedtoidentifyandquantifyphosphorylationchanges;however,phosphoproteinandphosphopeptideanalysisbyMSislimitedbymanyfactors,includingdigestionefficiency,lowstoiochiometry,lowabundance,hydrophilicity,poorionizationandpoorfragmentation.Asaresult,phosphopeptideenrichmentisessentialtosuccessfulMSanaly-sis.ThenewPierceFe-NTAPhosphopeptideEnrichmentKitiscompatiblewithourlysis,reduction,alkylation,anddigestionreagentsandwithThermoScientificPierceGraphiteSpinColumnstoprovideacompleteworkflowforphosphopeptideenrichment.
Toassessphosphopeptideenrichmentfromlysates,culturedU2-OScellsarrestedwithnocodazole(100ng/mL,25hours)werelysedwith6Mureain50mMTris,pH8.0containingThermoScientificHaltProteaseandPhosphataseInhibitorCocktail(Product#78440).ProteinconcentrationwasdeterminedwithThermoScientificPierce660nmProteinAssay(Product#22660).ProteinswerereducedwithThermoScientificBond-BreakerTCEPSolution,NeutralpH(Product#77720),alkylatedwithsingle-useiodoacetamide,(Product#90034)digestedovernightwithMS-gradetrypsin(Product#90055),anddesaltedwithThermoScientificHyperSep-C18Cartridges(Product#60108-305).Anequivalentof200μgofpeptidesweredriedanddissolvedin5%aceticacidorSigmaPhos-Select™Buffer.PhosphopeptideswereenrichedwithPierceFe-NTAPhosphopeptideEnrichmentKitorSigmaPhos-SelectReagentsandthendesaltedandconcentratedwithPierceGraphiteSpinColumns(Product#88302)accordingtoinstructions.
EnrichedphosphopeptidesampleswereanalyzedbyLC-MS/MS.ANanoLC™-2DHPLC(Eksigent)withaProteoPepIIC18Column(75μmIDx20cm,NewObjective)wasusedtoseparatepeptidesusinga4-40%gradientofsolvents(A:water,0.1%formicacid;B:acetonitrile,0.1%formicacid)at250nLperminutefor60minutes.PeptideswereidentifiedwithaThermoScientificLTQOrbitrapXLETDMassSpectrometerusingatopfourexperimentconsistingofhigh-resolutionMSfollowedbyacquisitionoffourMS/MSspectrausingtheCIDmodeoffragmentation.LC-MS/MSdatawereinterpretedwithMascot2.2(MatrixScience)andScaffold2.6(ProteomeSoftware).
ToachieverobustMSresults,enrichmentofphosphopeptidesamplesisessentialbecauseoflowstoichiometryandabundanceandpoorionizationrelativetononphosphorylatedpeptides.Wehavedevelopedanefficientmeanstoenrichphosphopeptidesfromcomplexsamples.ThenewThermoScientificPierceFe-NTASpinColumnseffectivelycapture,enrich,andrecoverphosphopeptides.Thesecolumnsenrichahigherpercentageofphosphopeptidesthanotherresinsandwithanoverallhighernumberoftotalanduniquephosphopeptides(Figure8andTable3).
0
10
20
30
40
50
60
70
Phos
phop
eptid
es (%
)
Thermo Scientific Pierce Fe-NTA Kit Sigma Phos-SelectReagent
Total Phosphopeptides
862
43090
178Unique Phosphopeptides
Figure 8. Our kit enriched a greater percentage of total and unique phospho-peptides from U2-OS cell lysate.Numbersrefertothetotalanduniquenum-berofphosphopeptidesidentifiedineachcondition.AsummaryofresultsislistedinTable3.
54 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Table 3. Average phosphopeptide enrichment results from duplicate experiments.§
Thermo Scientific Pierce Fe-NTA
Sigma Phos-Select
Totalphosphopeptides 862 430
Totalpeptides 1,753 1,665
Totaluniquepeptides 393 395
Totaluniquephosphopeptides 178 90
Totalphosphopeptides(%) 53 31
Uniquephosphopeptides(%) 50 27.5
§ Peptide summary results were exported from Scaffold and analyzed and summarized with Microsoft Excel® and Access®.
Multiplephosphorylatedaminoacidswithinapeptidecontributetothecomplexityofphosphopeptideanalysis.PierceFe-NTASpinColumnsenrichpeptideswiththreeormorephosphorylatedsitesandsignificantlyoutperformothercommerciallyavailablecolumns(Figure9).Phosphopeptideenrichmentgreatlyreducessamplecomplexityandenableseffectiveidentificationandcharacteriza-tionofphosphorylatedpeptidesbyMS(Figure10).
0
20
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60
80
100
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Phos
phop
eptid
es (%
)
Single Double
Phosphates per Peptide
Triple ≥4
Thermo Scientific Pierce Fe-NTA Kit110
29
132 12 1 0
Sigma Phos-Select Reagent
Figure 9. The Thermo Scientific Pierce Fe-NTA Phosphopeptide Enrichment Kit effectively captures phosphopeptides with multiple phosphates.
ThePierceFe-NTAPhosphopeptideEnrichmentKitcontainsdetailedinstructionsandallnecessarycomponentstoload,washandelutephosphopeptideswithinanhour.Thiskitiscompatiblewithsamplesdigestedinsolutionorafterin-geldigestionusingtheThermoScientificIn-gelTrypticDigestionKit(Product#89871).
a11+2H+1
y2 b4y3 b5y4 b6 y5 b7y6
b8y7 y8b9 y9b10
G E P N V S Y+80 I C+57 S RR S C+57 I Y+80 S V N P E G
m/z
0%
25%
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GSK3A_HUMANGlycogen Synthase Kinase-3 alpha
(R)GEPNVSyICSR(Y)
CDC2_HUMANCell division control protein 2 homolog
(K)IGEGTyGVVYK(G)
b3y2
b4y3
b5 y4 y5 b6 b7y6
b8 y7 b9
y8
y9b10y10
I G E G T Y+80 G V V Y KK Y V V G Y+80 T G E G I
Ordering Information Product #
Description
Pkg. Size
U.S. Price
88300 Pierce Fe-NTA Phosphopeptide Enrichment KitSufficient for 30 samples
Kit $279
Related Products88302 Pierce Graphite Spin Columns 30columns $105
Figure 10. Example MS/MS spectra from enriched phosphopeptides. Lowercaselettersindicatethepositionoftyrosinephosphorylationinenrichedpeptides.Panel A:Glycogensynthase-3alpha.Panel B:cdc2/cyclindependentkinase1.
Phosphopeptide Enrichment
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 55
Cell Surface Protein Isolation Kit
Convenient biotinylation and isolation of cell surface proteins for Western blot analysis.
TheThermoScientificPierceCellSurfaceProteinIsolationKitisacompletekitfortheconvenientbiotinylationandisolationofmammaliancellsurfaceproteins,specificallytargetingcellsurfaceproteinstotheexclusionofintracellularproteins.Thekitefficientlylabelsproteinswithaccessiblelysineresiduesandsufficientextracellularexposure.
Theisolationprocedureusesacell-impermeable,cleavablebiotinylationreagent(Sulfo-NHS-SS-Biotin)tolabelsurfaceproteinsatexposedprimaryamines.Cellsarethenharvestedandlysed,andthelabeledsurfaceproteinsareaffinity-purifiedusingThermoScientificNeutrAvidinAgaroseResin.Theisolatedcellsurfaceproteinscontainasmall,nonreactivetagoftheoriginallylabeledprimaryaminesbutarenolongerbiotinylated(biotinremainsboundtotheresin).
Highlights:• Isolates cell surface proteins–reducescomplexityoftotal
cellularprotein• Efficiently recovers labeled proteins–cleavablebiotinallowsfor
nearly100%recoveryofisolatedcellsurfaceproteins• Convenience–allreagentsareprovidedinonekit,alongwith
completeinstructionsforlabeling,celllysisandpurificationofcellsurfacemembraneproteins
• Western blotting applications–proteinsrecoveredinSDS-PAGEbufferareloadeddirectlyontopolyacrylamidegels
• Robust system–protocoldesignedfordiversecelllines,includingNIH3T3,HeLa,C6andA431
A. Cell Surface Proteins
EGFR
IGF-1Rβ
Hsp90 Calnexin
Integrin α5
Integrin β1
R F E R F E
R F E R F E
R F E R F E R F E R F E
R F E R F E
R F E R F E
B. Intracellular Proteins
+ – + –
+ –
+ –
+ –
+ –
Protein isolation is specific to cell surface proteins. PanelsareWesternblotresultsforknowncellsurfaceproteins (Panel A) andintracellularproteins(Panel B)fromHeLacellstestedwiththeCellSurfaceProteinIsolationKit.Plussymbol(+)denotesresultsforcellstreatedwiththeSulfo-NHS-SS-Biotinreagent;minussymbol(-)denotesresultsforcellsthatwerenottreatedwiththebiotinreagentbutwereotherwisecarriedthroughthekitprocedure.Lanesareno-sampleresin-control(R),flow-through(F)andeluted(E)frac-tions.Presenceoftargetcellsurfaceproteinsintheplus-Eandminus-Fconditionsindicatesuccessfulisolationwiththekit.PresenceofintracellularproteinsinFconditionofbothplusandminusconditionsindicatesthatthelabelingandpurificationprocedureisspecifictocellsurfaceproteins.
Ordering Information
Product # Description Pkg. SizeU.S. Price
89881 Cell Surface Protein Isolation KitSufficient reagents and accessories for eight experiments, each involving four T75 flasks of confluent cells.Includes:EZ-Link®Sulfo-NHS-SS-Biotin
QuenchingSolutionLysisBufferNeutrAvidinAgaroseWashBufferDithiothreitol(DTT)BupHPhosphateBufferedSalineBupHTrisBufferedSalineSpinColumnsandAccessories
Kit
8x12mgvials16mL4.5mL2.25mL34mL8x7.7mgmicrotubes2packs1pack
$348
Biotinylate cells
30 minutes at 4˚C
Transfer cell pellet to 1.5 ml tube
Lyse cells 30 minutes on ice
Isolate biotinylated proteins on NeutrAvidin® Agarose Resin
Perform electrophoresis
or other application
Wash gel then elute with SDS-PAGE sample buffer + 50 mM DTT
Harvest Cells
Quench Reaction
gel
B
SH
+
protein – SH
N
N
gel
N
B
S-S-protein
N
N Biotin NeutrAvidinBiotin-Binding Protein
1D gel
B
45
40
35
30
25
20
15
107.55.0
Protocol summary for the Thermo Scientific Pierce Cell Surface Protein Isolation Kit.
Protein Enrichment
56 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Glycoprotein Isolation Kits
Isolate glycoproteins from complex protein mixtures.
Twolectin-basedThermoScientificGlycoproteinIsolationKits,concanavalinA(ConA)andwheatgermagglutinin(WGA),allowisolationofglycoproteinsfromcomplexproteinmixtures,includingserum,tissueandculturedcelllysates,thusenablingdownstreamanalysis.ConAlectinrecognizesa-linkedmannoseandterminalglucoseresidues,whileWGAlectinselectivelybindstoN-acetylglucosamine(GlcNAc)groupsandtosialicacid.
Highlights:• High recovery–equivalentorgreaterglycoproteinrecoveryvs.
competitorkitsandlectinresins• Fast–glycoproteinpurificationinlessthanonehour• Versatile–isolateglycoproteinsfromvarioussampletypes;
e.g.,humanserumandcelllysate• Robust–lectindoesnotleachfromresinwhen
processingsample• Convenient–completekitcontainslectinresinsandspin
columnswithallnecessaryreagents• Compatible with Bradford-based protein assays–dialysisor
proteinprecipitationofrecoveredglycoproteinsisnotrequiredbeforeproteinassay
Ther
mo
Scie
ntifi
c
Ther
mo
Scie
ntifi
c
Supp
lier A
Supp
lier A
Supp
lier G
ConA
Supp
lier G
ConA
B.A.
Human Serum CHO Lysate
Glycoprotein isolation from human serum and cell lysate: performance comparison of kits using ConA resin. HumanserumandCHOlysatesampleswereprocessedwiththeThermoScientificGlycoproteinIsolationKit,ConAandwithothercommerciallyavailableConAresins.Anequivalentamountoftotalproteinwasappliedtoeachresin.ElutedglycoproteinfractionswerecomparedwithConAResinboiledinSDS-PAGEBuffertoreleaselectins.Allfractionswerenormalizedbyvolumeandresolvedon8-16%polyacrylamidegels.Gelsweresilver-stained.A.ElutedglycoproteinfractionsfromappliedhumanserumandB.elutedglycoproteinfractionsfromappliedCHOlysate.ArrowsidentifyproteinbandsthatresultfromConAleachingfromtheresinduringtheelutionprocess.
Com
petit
or A
Supp
lier A
Supp
lier A
B.A.
CHO LysateHuman Serum
Ther
mo
Scie
ntifi
c
Ther
mo
Scie
ntifi
c
Glycoprotein isolation from human serum and cell lysate: performance comparison of kits using WGA resin.HumanserumandCHOlysatesampleswereprocessedwiththeThermoScientificGlycoproteinIsolationKit,WGAandwithothercommerciallyavailableWGAresins.Anequivalentamountoftotalproteinwasappliedtoeachresin.Elutedglycoproteinfractionswerenormalizedbyvolumeandresolvedon8-16%polyacrylamidegels.A.ElutedglycoproteinfractionfromappliedhumanserumandB.elutedglycoproteinfractionfromappliedCHOlysate.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89804
Glycoprotein Isolation Kit, ConASufficient reagents to isolate glycoproteins with strong affinity for ConA from 10 samples of up to 640µL (1-1.5mg total protein) each.Includes:ConALectinResin,1.1mLresinsuppliedas
a50%slurryBinding/WashBuffer,5XStockSolutionElutionBufferColumnAccessoryPack,10SpinColumnswithCapsand20CollectionTubes
Kit
6.5mL5mL
$223
89805 Glycoprotein Isolation Kit, WGASufficient reagents to isolate glycoproteins with strong affinity for WGA from 10 samples of up to 640µL (1-1.5mg total protein) each.Includes:WGALectinResin,1.1mLresinsuppliedasa
50%slurryBinding/WashBuffer,5XStockSolutionElutionBufferColumnAccessoryPack,10SpinColumnswithCapsand20CollectionTubes
Kit
6.5mL5mL
$282
Protein Enrichment
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 57
Ubiquitin Enrichment Kit
Recover ubiquitin modified protein in <45 minutes.
Theubiquitinproteasomepathwayistheprincipalnon-lysosomalpathwaythatcontrolstheproteolysisofproteins.Thispathwayissignificantlyinvolvedinavarietyofcellularprocesses,includingDNArepair,transcriptionalregulation,signaltransduction,cellmetabolismandmorphogenesis.Differencesintotalubiquitinationortheubiquitinationofspecificproteinsaffectnumerouspathologicalconditions,includingmalignancies,certaingeneticdiseasesandneurodegenerativediseases.1
TheThermoScientificUbiquitinEnrichmentKitisolatespolyubiquitinproteinconjugatesfromculturedcellsandtissuesamples.TheenrichedfractionisanalyzedtodeterminetheamountofgeneralubiquitinconjugatespresentortoidentifyaspecificproteinbyWesternblotting.Theassayprotocolisfast,straightforwardandallowsisolationofpolyubiquitinatedproteinsandthefractionationofmonoubiquitinatedspeciesintheresinflow-through.TheUbiquitinEnrichmentKitoutperformsothersuppliers’kitsandprovidesacleanandspecificpreparationofproteinswhencomparedtoacontrolresin.
Highlights:•Fast–lessthan45minuteshands-ontime• Complete–includesallreagentsneededforubiquitin-modified
proteinenrichmentfromculturedcellsandtissuesamples,includingspincolumnsandubiquitinantibody
• Flexible–sampleincubationfrom2hourstoovernightallowsassaytobecompletedinseveralhoursorinlessthan30minutesafterovernightincubation
• Robust–compatiblewithallThermoScientificCellLysisSolutionsandstandardRIPAformulations
• Multiple-sample format–easilyprocesses1-15samplesconcurrently
20
40
5060
80100120
220
Thermo Scientific
KitSupplier
CAb-basedMethod
GSH Resin
H F E F E F E F E
kDa
The Thermo Scientific Ubiquitin Enrichment Kit recovers more ubiquitin-modified proteins than any other method. Epoxomicin-treatedHeLacelllysates(EHeLa,150μg)wereenriched.Afterelution,allsampleswerenormalizedtotheinitialload(EHeLaload).Theflow-throughandelutionobtainedusingtheThermoScientificUbiquitinEnrichmentKitareshownfirst.Theresultsusinganothersupplier’senrichmentkitareshownforcomparison(SupplierC,manufacturer’sinstructionsforthiskitwerefollowed).Additionally,theresultsobtainedusingananti-ubiquitinmonoclonalantibody-basedenrichmentscheme(antibody-based)andanegativecontrolresin(GSHresin)areshown.Theelutionfromeachresinshowstheamountofubiquitin-modifiedproteinthatwascapturedusingthatmethod.
Reference1.Ciechanover,A.(1998).Theubiquitin-proteasomepathway:onproteindeathandcelllife.
EMBO J.17(24),7151-1760.
Ordering Information
Product # Description Pkg. SizeU.S. Price
89899
Ubiquitin Enrichment KitContains sufficient materials for enriching up to 15 lysate samples containing ~0.15mg total protein per sample.Pack 1PolyubiquitinPositiveControl(1,000X),50μL,2mg/mLAnti-ubiquitinAntibody,50μLrabbitantiserumPack 2PolyubiquitinAffinityResin,300μL,suppliedasa25%slurryBindingCapacity:~1μgper20μLofslurryBupHTrisBufferedSalinePack,1ea.,makes500mLof0.025MTris,0.15MNaCl;pH7.2SpinColumnsandAccessories,18columnswithtopandbottomcaps
Kit $399
58 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Antibody Purification Overview
Antibodies specific for an antigen of interest are one of the most useful and important tools that biology researchers can possess. The production and use of specific antibodies as detection probes and purification ligands (i.e., immunotechnology) has revolutionized bioresearch and diagnostic technologies. Animalsimmunizedwithpreparedantigenswillproducespecificantibodiesagainsttheantigen.Whenpurifiedfromserumorhybridomacelllinesthatarepreparedfromtissueoftheimmunizedanimal,theantibodycanbeuseddirectly(orafterlabelingwithenzymeorfluorescenttags)toprobethespecificantigeninWesternblotting,ELISAoravarietyofotherapplications.Antibodiesaremostcommonlypurifiedbyoneoftwoaffinitypurificationmethods:generalimmunoglobulinpurification(pages58-65)orspecificantibodypurification(seepages26-35).SeealsoTable1.
General Purification of Immunoglobulins
Becauseantibodieshavepredictablestructure,includingrelativelyinvariantdomains,ithasbeenpossibletoidentifycertainproteinligandsthatarecapableofbindinggenerallytoantibodies,regardlessoftheantibody’sspecificitytoantigen.ProteinA,ProteinGandProteinLarethreebacterialproteinswhoseantibody-bindingpropertieshavebeenwellcharacterized.Theseproteinshavebeenproducedrecombinantlyandusedroutinelyforaffinitypurificationofkeyantibodytypesfromavarietyofspecies.AgeneticallyengineeredrecombinantformofProteinAandG,calledProteinA/G,isalsoavailable.Theseantibody-bindingproteinsareavailableimmobilizedtobeadedagaroseresin,ThermoScientificUltraLinkBiosupportandcoatedontomicroplates.
ProteinsA,G,A/GandLbindtoantibodiesatsitesotherthantheantigen-bindingdomain.Therefore,theseproteinscanbeusedinpurificationschemessuchasimmunoprecipitation.
ProteinsA,G,A/GandLhaveuniqueproperties,whichmakeeachonesuitablefordifferenttypesofantibodytargets(e.g.,antibodysubclassoranimalspecies).ItisimportanttorealizethatuseofProteinA,GorLresultsinpurificationofgeneralimmunoglobulinfromacrudesample.Dependingonthesamplesource,antigen-specificantibodymayaccountforonlyasmallportionofthetotalimmunoglobulininthesample.Forexample,generallyonly2-5%oftotalIgGinmouseserumisspecificfortheantigenusedtoimmunizetheanimal.
Table 1. Antibody Purification Methods.
Purification Type Description Available Thermo Scientific Support
General Negativeselection Removalofallnon-immunoglobulinproteinsfromaserumsample
MelonGel
IgGenrichment ImmobilizedglobulinbindingproteinstoselectivelyremoveIgGfromaserumsample
ImmobilizedProteinA
ImmobilizedProteinG
ImmobilizedProteinA/G
ImmobilizedProteinL
IgGenrichment Thiophilicadsorption ThiophilicAdsorbent
IgMenrichment UseMannanbindingproteintoselectivelyisolateIgM ImmobilizedMannanBindingProtein
IgAenrichment UseJacalin,aD-galactosebindinglectin,toselectivelyisolateIgA
ImmobilizedJacalin
IgYenrichment Delipidationandprecipitationfromeggyolks IgYPrecipitationReagent
Specific (seepage26)
Affinitypurification Createanaffinitycolumnwiththeantigenusedtoimmunizetheanimal.
NHS-esterActivatedAgarose-forimmobilizingantigensviaprimaryamines
AminoLinkPlusAgarose-forimmobilizingantigensviaprimaryamines
SulfoLinkResin-forimmobilizingantigensviareducedsulfhydryls
CarboxyLinkResin-forimmobilizingantigensviacarboxylicacids
Antibody Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 59
w = weak binding, m = medium binding, s = strong binding, nb = no binding, – means information not available * Data represent a summary of binding properties reported in the literature. Inevitably some discrepancies exist among reported values as a result of differences in binding
buffer conditions and form of the proteins used. † Binding will occur only if the appropriate kappa light chains are present. Antibodies lambda light chains will not bind, regardless of their class and subclass.
Protein A Protein G Protein A/G Protein L†ThiophilicAdsorbent
HumanIgG s s s s m
MouseIgG s s s s s
RabbitIgG s s s w m
GoatIgG w s s nb s
RatIgG w m m s s
SheepIgG w s s nb s
CowIgG w s s nb s
GuineaPigIgG s w s – s
HamsterIgG m m m s –
PigIgG s w s s s
HorseIgG w s s – s
DonkeyIgG m s s – –
DogIgG s w s – s
CatIgG s w s – s
MonkeyIgG(Rhesus)
s s s – s
ChickenIgY nb nb nb nb m
HumanIgM w nb w s m
HumanIgE m nb m s –
HumanIgD nb nb nb s –
HumanIgA w nb w s m
HumanIgA1 w nb w s m
HumanIgA2 w nb w s m
HumanIgG1 s s s s m
Protein A Protein G Protein A/G Protein L†ThiophilicAdsorbent
HumanIgG2 s s s s m
HumanIgG3 w s s s m
HumanIgG4 s s s s m
HumanFab w w w s m
HumanScFv w nb w s m
MouseIgG1 w m m s s
MouseIgG2a s s s s s
MouseIgG2b s s s s s
MouseIgG3 s s s s s
RatIgG1 w m m s s
RatIgG2a nb s s s s
RatIgG2b nb w w s s
RatIgG2c s s s s s
CowIgG1 w s s nb s
CowIgG2 s s s nb s
SheepIgG1 w s s nb s
SheepIgG2 s s s nb s
GoatIgG1 w s s nb s
GoatIgG2 s s s nb s
HorseIgG(ab) w nb w – s
HorseIgG(c) w nb w – s
HorseIgG(T) nb s s – s
MouseIgM nb nb nb s m
Immobilized Protein L, Protein A, Protein G and Protein A/G
Weofferthesepopularantibody-bindingproteinsimmobilizedonseveraldifferentresins,beadsandplatesforuseinimmunoaffinitypurificationtechniques.Allfourproteins(A,G,A/GandL)areavailableaspurifiedrecombinantsimmobilizedtocrosslinked6%beadedagarose.Thisisthetraditionalformathistoricallyusedforsmall-scalecolumnpurificationandimmunoprecipitationmethods.Ouragaroseresinsdifferfromthosetypicallyofferedbyothersuppliersinthatourimmobilizationmethodismorestableandresultsinlessnonspecificbinding.Wealsooffer“Plus”versionsoftheProteinA,G,A/GandLagaroseresins,whichcontaintwice
theamountofproteinpermilliliterofresinandprovidefornearlytwicetheantibodybindingcapacity.
ProteinA,G,A/GandLarealsoavailableimmobilizedtoUltraLink®Biosupport,anextremelydurable,polyacrylamide-basedresinwithverylownonspecificbindingcharacteristics.TheUltraLinkFormatisaperfectsupportforworkingwithlargevolumesamplesinlarge-scalepurificationmethodsrequiringfastflowandhighpressure.
TheinteractionbetweenthevariousproteinsandIgGisnotequivalentforallspeciesorallantibodysubclasses.Thetablesonthefollowingpagewillhelpyoudecidewhichaffinityproteinisbestforyourapplication(Tables2and3).
Table 2. Characteristics of immunoglobulin-binding proteins.
Recombinant Protein L
Native Protein A
Recombinant Protein A
Recombinant Protein G
Recombinant Protein A/G
ProductionSource E. coli S. aureus E. coli E. coli E. coli
MolecularWeight 35,800 46,700 44,600 21,600 50,460
NumberofBindingSitesforIgG 4 4 4 2 6
Albumin-BindingSite No No No No No
OptimalBindingpH 7.5 8.2 8.2 5 5-8.2
Bindsto VLK FC FC FC FC
Table 3. Binding characteristics of immunoglobulin-binding proteins and Thermo Scientific Thiophilic Adsorbent.*
60 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Protein A
Protein A Beads – Quick Reference
ProteinA NativeproteinpurifiedfromStaphylococcus aureus(46.7kDa;fourIgG-bindingsites)
Specificity(Table3)
BestforpolyclonalIgGfromrabbit,pig,dog,catserum;poorforMouseIgG1,humanIgG3,rat,goat,cow
SupportsOffered
Crosslinked6%beadedagaroseUltraLinkBiosupportTrisacrylGF-2000ResinMagneticparticles(1-4μm)
PackageFormats
Resinslurries(3sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NAb™IgGPurificationKits(2sizes)
Storage 4°C,donotfreeze
Capacity ProteinA:12-19mghumanIgG/mLresinProteinAPlus:>35mghumanIgG/mLresin;
16-17mgmouseIgG/mLresinProteinATrisacrylResin:>15mghumanIgG/mLresinProteinAUltraLinkResin:>16mgofhumanIgG/mLresinProteinAPlusUltraLinkResin:>30mghumanIgG/mLresinProteinAMagnaBindBeads:>0.2mgrabbitIgG/mLbeadsProteinACoatedPlates:~4pmolrabbitIgG/wellRecombinantProteinA:≥12mghumanIgG/mLresinusingthe
IgGBufferSystem
Ordering Information
Product #
Description
Pkg. Size
U.S. Price
20333 Protein A Agarose 5mL $ 337
20334 Protein A Agarose 25mL $1050
20356 Protein A Columns 5x1mL $ 385
44667 Protein A IgG Purification Kit Kit $ 515
20338 Protein A Trisacryl Resin 5mL $ 350
53139 Protein A UltraLink Resin 5mL $ 340
22810 Protein A Plus Agarose 1mL $ 120
22811 Protein A Plus Agarose 5mL $ 400
22812 Protein A Plus Agarose 25mL $1350
89924 Chromatography Cartridges, Protein A 2x1mL $ 170
89925 Chromatography Cartridge, Protein A 1x5mL $ 335
89952 NAb Protein A Plus Spin Columns 10x0.2mL $ 222
89956 NAb Protein A Plus Spin Columns 5x1mL $ 425
89960 NAb Protein A Plus Spin Column 1x5mL $ 400
89948 NAb Protein A Plus Spin Purification Kit Kit $ 270
89978 NAb Protein A Plus Spin Purification Kit Kit $ 265
53142 Protein A UltraLink Plus Resin 5mL $ 399
45202 Protein A Spin Plate 1plate $ 188
21348 MagnaBind Protein A Beads 5mL $ 325
15130 Protein A, Clear 96-Well Plates 5plates $ 160
15132 Protein A, Clear 8-Well Strip Plates 5plates $ 178
15154 Protein A, White 96-Well Plates 5plates $ 178
15155 Protein A, Black 96-Well Plates 5plates $ 178
20365 Recombinant Protein A Agarose 5mL $ 275
20366 Recombinant Protein A Agarose 25mL $1001
Protein G
Protein G Beads – Quick Reference
ProteinG RecombinantproteinexpressedinE. coli(21.6kDa;twoIgGbindingsites)
Specificity(Table3)
BestforIgGfrommouse,human,cow,goatandsheep;poorforguineapig,pig,dogandcat
SupportsOffered
Crosslinked6%beadedagaroseUltraLinkBiosupportMagneticparticles(1-4μm)
PackageFormats
Resinslurries(3sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NabIgGPurificationKits(2sizes)
Storage 4°C,donotfreeze
Capacity ProteinG:11-15mghumanIgG/mLresinProteinGUltraLinkResin:>20mgofhumanIgG/mLresinProteinGPlus:>20mghumanIgG/mLresinProteinGUltraLinkResin:>20mgofhumanIgG/mLresinProteinGPlusUltraLinkResin:>25mghumanIgG/mLresinMagnaBindProteinGBeads:>0.2mgrabbitIgG/mLbeadsProteinGCoatedPlates:~2pmolrabbitIgG/wellProteinGPlus:>20mghumanIgG/mLresinProteinGPlusUltraLinkResin:>25mghumanIgG/mLresin
Ordering Information
Product #
Description
Pkg. Size
U.S. Price
20398 Protein G Agarose 2mL $ 212
20399 Protein G Agarose 10mL $ 760
20397 Protein G Agarose 25mL $1654
89926 Pierce Chromatography Cartridges, Protein G 2x1mL $ 190
89927 Pierce Chromatography Cartridge, Protein G 1x5mL $ 390
89953 NAb Protein G Spin Columns 10x0.2mL $ 251
89957 NAb Protein G Spin Columns 5x1mL $ 455
89961 NAb Protein G Spin Columns 1x5mL $ 425
89949 NAb Protein G Spin Purification Kit Kit $ 310
89979 NAb Protein G Spin Purification Kit Kit $ 368
21193 Pierce Recombinant Protein G 5mg $ 300
53125 Protein G UltraLink Resin 2mL $ 270
53126 Protein G UltraLink Resin 10mL $ 910
53127 Protein G UltraLink Columns 2x2mL $ 540
45204 Protein G Spin Plate 1plate $ 209
22851 Protein G Plus Agarose 2mL $ 260
22852 Protein G Plus Agarose 10mL $ 895
53128 Protein G Plus UltraLink Resin 2mL $ 335
21349 MagnaBind Protein G Beads 5mL $ 325
15131 Protein G, Clear 96-Well Plates 5plates $ 178
15133 Protein G, Clear 8-Well Strip Plates 5plates $ 195
15156 Protein G, White 96-Well Plates 5plates $ 195
15157 Protein G, Black 96-Well Plates 5plates $ 195
Antibody Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 61
Protein A/G
Protein A/G Beads – Quick Reference
ProteinA/G RecombinantproteinexpressedinE. coli(50.5kDa;sixIgG-bindingsites)
Specificity(Table3)
BestforpolyclonalIgGfrommanyspecies;poorforindividualsubclassesthathavehighestaffinityforProteinAorProteinG.
SupportsOffered
Crosslinked6%beadedagaroseUltraLinkBiosupport
PackageFormats
Resinslurries(3sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NAbIgGPurificationKits(2sizes)
Storage 4°C,donotfreeze
Capacity ProteinA/G:>7mghumanIgG/mLresinProteinA/GUltraLinkResin:>20mghumanIgG/mLresinProteinA/GPlus:>50mghumanIgG/mLresinProteinA/GPlusonUltraLinkSupport:>28mghumanIgG/mLresinProteinA/GCoatedPlates:~5pmolrabbitIgG/well
Ordering Information Product #
Description
Pkg. Size
U.S. Price
15138 Protein A/G, Clear 96-Well Plates 5plates $ 196
20421 Protein A/G Agarose 3mL $ 315
20422 Protein A/G Agarose 15mL $1150
89930 Pierce Chromatography Cartridges, Protein A/G 2x1mL $ 299
89931 Pierce Chromatography Cartridge, Protein A/G 1x5mL $ 399
89954 NAb Protein A/G Spin Columns 10x0.2mL $ 260
89958 NAb Protein A/G Spin Columns 5x1mL $ 460
89962 NAb Protein A/G Spin Column 1x5mL $ 435
89950 NAb Protein A/G Spin Purification Kit Kit $ 336
89980 NAb Protein A/G Spin Purification Kit Kit $ 375
53132 Protein A/G UltraLink Resin 2mL $ 270
53133 Protein A/G UltraLink Resin 10mL $ 910
21186 Pierce Recombinant Protein A/G 5mg $ 303
20423 Protein A/G Plus Agarose 2mL $ 340
53135 Protein A/G Plus on UltraLink Support 2mL $ 367
Protein L
Protein L Beads – Quick Reference
ProteinL RecombinantproteinexpressedinE. coli(35.8kDa;fourIgG-bindingsites)
Specificity(Table3)
Bestforhumanormousemonoclonalantibodiesknowntohaveappropriatekappalightchains;poorforgeneral-purpose(polyclonal)IgGpurification
SupportsOffered
Crosslinked6%beadedagarose
PackageFormats
Resinslurries(2sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NAbIgGPurificationKits(2sizes)
Storage 4°C,donotfreeze
Capacity ProteinL:5-10mghumanIgG/mLresinProteinLPlus:>10-20mghumanIgG/mLresin
Ordering Information
Product #
Description
Pkg. Size
U.S. Price
20510 Protein L Agarose 2mL $248
20512 Protein L Agarose 10mL $950
89928 Pierce Chromatography Cartridges, Protein L 2x1mL $190
89929 Pierce Chromatography Cartridge, Protein L 1x5mL $375
89955 NAb Protein L Spin Columns 10x0.2mL $270
89959 NAb Protein L Spin Columns 5x1mL $489
89963 NAb Protein L Spin Column 1x5mL $459
89951 NAb Protein L Spin Purification Kit Kit $365
89981 NAb Protein L Spin Purification Kit Kit $410
21189 Pierce Recombinant Protein L 1mg $121
20520 Protein L Plus Agarose 2mL $365
15190 Protein L, Clear 96-Well Plates 5plates $191
For complete product details,
visit www.thermoscientific.com/pierce or request the Antibody Purification
Handbook (1601974)
Version 2
Thermo Scientific Pierce Antibody Production and Purification Technical Handbook
62 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
IgG Binding and Elution Buffers for Protein A, G, A/G and L
Binding and Elution Steps in Affinity PurificationAffinitypurificationproceduresinvolvinginteractionofanantibodywithitsantigengenerallyusebindingbuffersatphysiologicpHandionicstrength.However,manyantibodypurificationmethodsdonotusetheantibody-antigeninteraction;rather,theyinvolvebindingofantibodiesbyimmobilizedligandsthatarenottheantigen.Insuchcases,optimalbindingconditionsaredeterminedbytheuniquepropertiesoftheantibody-ligandinteraction,whichmaybedifferentfromphysiologicpHandionicstrength.
Oncethebindinginteractionoccurs(i.e.,theantibodyis“captured”bytheimmobilizedligand),thesupportiswashedwithadditionalbuffertoremovenonboundcomponentsofthesample.Finally,
elutionbufferisaddedtobreakthebindinginteractionandreleasethetargetmolecule,whichisthencollectedinitspurifiedform.ElutionbuffercandissociatebindingpartnersbyextremesofpH(loworhigh),highsalt(ionicstrength),theuseofdetergentsorchaotropicagentsthatdenatureoneorbothofthemolecules,removalofabindingfactor,orcompetitionwithacounterligand.Inmostcases,subsequentdialysisordesaltingisrequiredtoexchangethepurifiedproteinfromelutionbufferintoamoresuitablebufferforstorageoruse.
ThermoScientificPierceIgGBindingandElutionBuffershavebeenoptimizedtoprovidethehighestpossibleefficiencyofIgGbindingandelutionusingimmobilizedProteinA,ProteinGandProteinA/G.Useofotherbufferformulationsmaysignificantlyalternotonlythebindingcapacitybutalsothevolumesofwashbufferrequiredtoensuregoodpurification.
Binding capacities with different Thermo Scientific Buffers expressed asmg of IgG bound per 2mL of resin.
Immobilized Protein A Immobilized Protein G Immobilized Protein A/G
Serum Sample0.1 M Tris•HCl
pH 8.0Pierce Protein A Binding Buffer
0.1 M Tris•HCl pH 8.0
Pierce Protein G Binding Buffer
0.1 M Tris•HCl pH 8.0
Pierce Protein A Binding Buffer
Rabbit 17.81 33.19 21.51 27.75 13.89 19.61
Sheep 2.15 10.64 25.53 33.33 9.83 15.71
Bovine 6.16 22.76 31.72 48.10 15.13 22.06
Mouse 5.25 7.15 5.65 15.05 4.32 11.49
Rat 4.99 8.30 8.43 11.80 5.20 6.66
Horse 6.25 16.50 36.19 21.46 14.88 17.12
Dog 35.77 22.27 13.38 20.55 21.96 24.60
Chicken 0.91 1.21 1.63 7.27 1.21 4.10
Pig 29.61 24.83 21.25 27.51 19.24 29.48
Human 19.88 25.53 11.68 23.59 9.92 17.67
Ordering Information Product #
Description
Highlights
Pkg. Size
U.S. Price
54200 Protein A/G IgG Binding Buffer •EnsuresmaximumrecoveryofIgGfromimmobilizedProteinA/G 240mL $ 37
2100121007
Protein A IgG Binding Buffer •High-yieldisolationofMouseIgG1usingProteinAcolumns•Premixedandeasytouse
1L3.75L
$ 70$195
21019 21011
Protein G IgG Binding Buffer •EnsuresmaximumrecoveryofIgGfromimmobilizedProteinG 1L3.75L
$ 66$194
2100421009
IgG Elution Buffer •High-yieldisolationofIgGfromImmobilizedProteinAandProteinG 1L3.75L
$ 65$165
2102021012
Gentle Ag/Ab Binding Buffer pH 8.0 •Speciallyformulatedandprefiltered•Eliminatesuseofharshacidicelutionconditions
1L3.75L
$103$197
210302102721013
Gentle Ag/Ab Elution Buffer pH 6.6 •Speciallyformulatedforneutralelutions•Notcompatiblewithphosphatebuffers
100mL500mL3.75L
$ 98$ 90$274
21016 IgM Binding Buffer •SpeciallyformulatedforoptimalbindingofmouseIgM 800mL $ 77
21017 IgM Elution Buffer •SpeciallyformulatedforoptimalrecoveryofmouseIgM 500mL $ 63
21018 MBP Column Preparation Buffer •SpeciallyformulatedforusewithImmobilizedMBPandIgMPurificationKit 50mL $ 33
21034 Mouse IgG1 Mild Elution Buffer •SeparateIgG1fromotherIgGsubclasses 500mL $ 80
21033 Mouse IgG1 Mild Binding and Elution Buffer KitIncludes:PierceProteinAIgGBindingBuffer MouseIgG1MildElutionBuffer PierceIgGElutionBuffer
•CompletekittoallowmouseIgG1tobeseparatedfromothermouseIgGsubclasses
Kit1L500mL1L
$255
Antibody Purification
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 63
Thermo Scientific Melon Gel Purification Products
Melon™GelProductsprovideanexcitingnewapproachtopurifyingmonoclonalandpolyclonalantibodiesfromserum,tissueculturesupernatantandascitesfluid.MelonGelworkswithantibodiesfromavarietyofspeciesandsubclasses,manyofwhichdonotpurifyefficientlywithProteinAorProteinG.BecauseMelonGelisnotabind-and-releasesupport,itisextremelyfastandgentletoyourantibodies,resultinginantibodypreparationsofhighpurityandhighactivity!
How does it work? MelonGelcontainsaproprietaryligandthatretainsmostproteinfoundinserum,ascitesandculturesupernatants,whileallowingIgGtopassthroughthesupportandbecollectedintheflow-throughfraction.TheresultingrecoveryandpurityoftheIgGisolatedbythismethodrivalsthatobtainedfromthesamesamplesusingbind-and-releasesupportssuchasProteinAorProteinG.
Highlights:• Simple, one-step protocol–notediousbinding,washing,and
multipleelutionsteps• Rapid purification–purifiesantibodiesfromserumfourtosix
timesfasterthanProteinAorGmethods• High recovery and purity–antibodiesfrommanyspecies
arerecoveredwithgreaterthan90%yieldandgreaterthan80%purity
• Robust purification–workswithawiderangeofantibodiesincludingmanythatdonotpurifywellonProteinAorProteinG
• Gentle purification–noharshelutionconditionsmeansantibod-iesretainmoreactivity
• Reusable support–MelonGelSupportcanbeusedformultipleantibodypurifications
• Available in various formats–spincolumns,purificationkitsandchromatographycartridgesforantibodypurificationfromserum,ascitesandculturesupernatant
IgG purification performance of the Thermo Scientific Melon Gel System, Protein A and Protein G.
Source Melon Gel Protein A Protein G
Human H H H
Mouse H H H
Rabbit H H H
Rat H L M
Goat H L M
Cow M L H
Sheep M L H
Horse H L H
GuineaPig H H L
Pig H H L
Chicken N N N
Hamster H M M
Donkey H M H
H = high recovery, M = medium recovery, L = low recover, N = no recovery
Ordering Information Product #
Description
Pkg. Size
U.S. Price
45206 Melon Gel IgG Spin Purification Kit Kit $ 243
45212 Melon Gel IgG Spin Purification Kit Kit $ 507
45214 Melon Gel Monoclonal IgG Purification Kit Kit $1106
45216 Saturated Ammonium Sulfate Solution 1L $ 147
45219 Ascites Conditioning Reagent 5mL $ 35
89932 Pierce Chromatography Cartridges, Melon Gel 2x1 $ 100
89933 Pierce Chromatography Cartridges, Melon Gel 1x5 $ 201
45208 Melon Gel Spin Kit Plate 2plates $ 311
89972 Melon Gel Purification Buffer 1pack $ 13
89973 Melon Gel Regenerant 1pack $ 10
For more data using Melon Gel and for complete
product information, please request the Antibody Purification Technical Handbook (1601974) or visit
www.thermoscientific.com/pierce
64 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Thiophilic Gel Antibody Purification
Thiophilicadsorptionisalow-cost,efficientalternativetoammoniumsulfateprecipitationforimmunoglobulinpurificationfromcrudesamples.Ammoniumsulfateprecipitationmustbefollowedbyseveraladditionalstepstocompletelyremovecontaminantsincrudesamples.Thiophilicadsorptionisasimple,rapid,one-stepmethodforantibodypurificationfromserum,ascitesortissueculturesupernatant.
Thiophilicadsorptionisahighlyselectivetypeoflyotropicsalt-promotedprotein:ligandinteractionphenomenonthathasbeenstudiedextensivelybyPorathandco-workersandotherresearchers.1Thisinteractionistermedthiophilicbecauseitdistinguishesproteinsthatrecognizeasulfonegroupincloseproximitytoathioether.Thiophilicadsorptionincorporatespropertiesofbothhydrophobicandhydrophilicadsorption.However,incontrasttostrictlyhydrophobicsystems,thiophilicadsorptionisnotstronglypromotedbyhighconcentrationsofsodiumchloride.Instead,thiophilicadsorptionispromotedbyincreasedconcentrationsofwater-interacting,non-chaotropicsaltssuchaspotassiumandammoniumsulfate.
ThermoScientificPierceThiophilicAdsorbentis6%beadedagarosemodifiedtocontainsimplesulfone/thioethergroups(seestructureatright).OurThiophilicAdsorbenthasahighbindingcapacity(20mgofimmunoglobulinpermLofresin)andbroadspecificitytowardimmunoglobulinsderivedfromvariousanimalspecies.Notably,thiophilicadsorptionisoneoffewmethodsavailableforpurificationofIgYfromchicken(seealsosubsequentdiscussionofIgYpurification).Amonghumanserumproteins,immunoglobulinsanda2-macroglobulinsarepreferentiallyboundbyourThiophilicAdsorbent.2
PurificationusingPierceThiophilicAdsorbentresultsingoodproteinrecoverywithexcellentpreservationofantibodyactivity.Samplepreparationrequirestheadditionof0.5Mpotassiumsulfatetotheserum,ascitesorculturefluid.GreaterspecificityforimmunoglobulinsisobtainedifthesampleisbufferedatpH8.0.Thegentleelutionconditions(e.g.,50mMsodiumphosphate,pH7-8)yieldconcentrated,essentiallysalt-free,highlypurifiedimmunoglobulinsatnearneutralpH.
Afteruse,ourThiophilicAdsorbentcanberegeneratedbytreatmentwithguanidine•HCl.OurdataindicatethattheAdsorbentcolumncanbeusedatleast10timeswithoutsignificantlossofbindingcapacity.
OS
SOH
OO
Aga
rose
Bea
d
Structure of Thermo Scientific Pierce Thiophilic Adsorbent.
References1.Porath,J.,et al. (1985).FEBS Lett.185,306-310.2.Belew,M.,et al. (1987).J. Immunol. Method102,173-182.3.Hutchens,T.W.andPorath,J.(1987).Biochemistry26,7199-7204.4.Lihme,A.andHeegaard,P.M.H.(1990).Anal. Biochem.192,64-69.5.Unpublishedinternaldocuments.
Thermo Scientific Pierce Thiophilic Adsorbent and Purification KitEconomical purification of mouse antibodies from ascites fluid.
Highlights:•BindstoFabandF(ab´)2fragments•BindstoScFv1•High-capacity(20mg/mL),goodproteinrecoveryandretention
ofantibodyfunction•Broadspecificitytowardimmunoglobulinsderivedfromvarious
animalspecies(seeTable4)•BindschickenIgY(alsoIgG)•Simple,rapid,one-steppurificationformonoclonalantibodies
fromascites;easytoscaleup•Usedtoenrichtheimmunoglobulinfractionfromserumortissue
culturesupernatant•Efficientalternativetoammoniumsulfateprecipitationfor
enrichingantibodiesfromcrudesamples•Gentleelutionconditionsyieldconcentrated,salt-free
immunoglobulinatnearneutralpH•Highdegreeofpurity
Table 4. Binding characteristics of Thermo Scientific Pierce Thiophilic Adsorbent.
Species
Total A280 Bound from 1mL Serum
% Purity by HPLC
Human 4.8 70
Mouse 8.6 63
MouseIgG1 11.6 92
MouseIgG2a 9.3 88
MouseIgG2b 9.8 97
MouseIgG3 10.7 94
Rat 13.0 79
Bovine 17.9 90
Calf 11.1 89
Chicken 5.2 76
Dog 12.2 91
Goat 17.3 92
GuineaPig 11.1 71
Horse 13.0 93
Pig 21.1 90
Rabbit 6.7 84
Sheep 12.3 89 Reference1.Schulze,R.A.,et al.(1994).Anal. Biochem.220,212-214.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20500 Pierce Thiophilic Adsorbent 10mL $ 92
44916 Pierce Thiophilic Purification KitIncludes:ThiophilicAdsorbentColumns BindingBuffer ElutionBuffer ColumnStorageBuffer(2X) Guanidine•HClCrystals ColumnExtenders
Kit4x3mL1,000mL1,000mL100mL230g
$443
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 65
IgM Purification
Immobilized Mannan Binding ProteinTodevelopaneffectiveaffinitymatrix,ourscientistsexaminedC1qandanothersimilarlystructuredprotein,mannanbindingprotein(MBP).SerumMBP,likeC1q,iscapableofinitiatingcarbohydrate-mediatedcomplementactivation.MBPisamannoseandN-acetylglucosamine-specificlectinfoundinmammaliansera,andithasconsiderablestructuralhomologytoC1q.8MBPsubunitsareidentical,eachwithmolecularmassofapproximately31kDa(C1qhassixeachofthreedifferentpolypeptidesubunitsofmolecularmass24-28kDa).StudiesinourlabsshowthatMBPdoesnotbindF(ab´)2andFab.
Wehavedevelopedaneasy-to-useThermoScientificPierceImmobilizedMannanBindingProteinandBufferSystemtopurifyIgM.ItismosteffectiveforpurifyingmouseIgMfromascites.PurifiedIgMcanbeobtainedfromasinglepassovertheaffinitycolumn.HumanIgMwillbindtothesupport,albeitwithslightlylowercapacity,andyieldaproductatleast88%pureasassessedbyHPLC.ThepurificationofIgMfromotherspeciesandmouseserumhasnotyetbeenoptimized.
Immobilized MBP and IgM Purification KitEasy IgM purification with guaranteed 88% pure mouse IgM!
0 10 20 30 40 50
0
20
40
60
80
100
Time (min.)
Abs
orba
nce
at 2
80 n
m(p
erce
nt o
f max
.)
IgM
Demonstration of the high purity of MBP-purified IgM from mouse ascites. Theboundmaterialfrommouseasciteswaselutedfromthe5mLMBPcolumnasdescribedintheStandardProtocol.Thehighest280nmabsorbingfractionfromtheelutionwaschromatographedusingtheconditionsdescribedintheinstructions.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
22212 Immobilized Mannan Binding ProteinCapacity:~1mgIgM/mLofresin
10mL $936
44897 IgM Purification Kit Kit $610
21016 IgM Binding Buffer 800mL $ 77
21017 IgM Elution Buffer 500mL $ 63
21018 MBP Column Preparation Buffer 50mL $ 33
53123 UltraLink Immobilized Mannan Binding ProteinCapacity:>0.75mgIgM/mLofresin
5mL $493
IgA Purification
Human IgA PurificationJacalinisana-D-galactosebindinglectinextractedfromjack-fruitseeds(Artocarpus integrifolia).Thelectinisaglycoproteinofapproximately40kDacomposedoffouridenticalsubunits.JacalinimmobilizedonsupportssuchasagarosehasbeenusefulforthepurificationofhumanserumorsecretoryIgA1.IgAcanbeseparatedfromhumanIgGandIgMinhumanserumorcolostrum.1IgDisreportedtobindtojacalin.2ImmobilizedjacalinisalsousefulforremovingcontaminatingIgAfromIgGsamples.
BindingofIgAtoimmobilizedjacalinoccursatphysiologicpHandionicstrength,asinphosphatebufferedsaline(PBS).ElutionofboundIgAoccurswithcompetitorligand(e.g.,0.1Mmelibioseor0.1Ma-D-galactose)inPBS.Weofferimmobilizedjacalinoncrosslinked6%agarose.
References1.Roque-Barreira,M.C.andCampos-Neto,A.(1985).J. Immunol. Method134(30),1740-1743.2.Aucouturier,P.,et al.(1987).Mol. Immunol.24(5),503-511.
Immobilized JacalinIdeal for human IgA purification.
Highlights:•IdealforpreparinghumanIgAthatisfreeofcontaminatingIgG•FoundtobindhumanIgA1,butnothumanIgA2–usefulforsepa-
ratingthetwosubclasses
ReferencesKumar,G.S.,et al.(1982).J. Biosci.4,257-261.Roque-Barreira,M.C.andCampos-Neto,A.(1985).J. Immunol.134,1740-1743.Mestecky,J.,et al.(1971).J. Immunol.107,605-607.VanKamp,G.J.(1979).J. Immunol. Method27, 301-305.Kondoh,H.,et al.(1986).J. Immunol. Method88,171-173.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20395 Immobilized JacalinCapacity:1-3mghumanIgA/mLofresinSupport:Crosslinked6%beadedagaroseLoading:4.5mgofjacalin/mLofresin
5mL $201
Antibody Purification
66 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Biotin
Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells. The valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules.Oncebiotinisattachedtoamolecule,themoleculecanbeaffinity-purifiedusinganimmobilizedversionofanybiotin-bindingprotein.Alternatively,abiotinylatedmoleculecanbeimmobilizedthroughinteractionwithabiotin-bindingprotein,thenusedtoaffinity-purifyothermoleculesthatspecificallyinteractwithit.Weofferbiotin-labeledantibodiesandanumberofotherbiotinylatedmolecules,aswellasabroadselectionofbiotinylationreagentstolabelanyprotein.
O
BiotinMW 244.3
S H
H
Valeric Acid Side Chain
H
H
O
HO
Biotin-Binding Proteins
Avidin–Theextraordinaryaffinityofavidinforbiotinallowsbiotin-containingmoleculesinacomplexmixturetobediscretelyboundwithavidin.Avidinisaglycoproteinfoundintheeggwhiteandtissuesofbirds,reptilesandamphibians.Itcontainsfouridenti-calsubunitshavingacombinedmassof67,000–68,000daltons.Eachsubunitconsistsof128aminoacidsandbindsonemoleculeofbiotin.Theextentofglycosylationonavidinishigh;carbohy-drateaccountsforabout10%ofthetotalmassofthetetramer.Avidinhasabasicisoelectricpoint(pI=10–10.5)andisstableoverawiderangeofpHandtemperature.Extensivechemicalmodificationhaslittleeffectontheactivityofavidin,makingitespeciallyusefulforproteinpurification.However,becauseofitscarbohydratecontentandbasicpI,avidinhasrelativelyhighnonspecificbindingproperties.
Streptavidin–Anotherbiotin-bindingproteinisstreptavidin,whichisisolatedfromStreptomyces avidiniiandhasamassof75,000daltons.Incontrasttoavidin,streptavidinhasnocarbohydrateandhasamildlyacidicpI(5.5).ThermoScientificPierceStreptavidinisarecombinantformhavingamassof53,000daltonsandanear-neutralpI.Streptavidinismuchlesssolubleinwaterthanavidin.Thereareconsiderabledifferencesinthecompositionofavidinandstreptavidin,buttheyareremarkablysimilarinotherrespects.Streptavidinisalsoatetramericprotein,witheachsubunitbindingonemoleculeofbiotinwithaffinitysimilartothatofavidin.Guanidiniumchloridewilldissociateavidinandstreptavidinintosubunits,butstreptavidinismoreresistanttodissociation.StreptavidincontainsanRYDsequencesimilartotheRGDsequencethatbindscellsurfacereceptors.TheRYDsequencecancausebackgroundinsomeapplications.
NeutrAvidin Protein–Wealsoofferadeglycosylatedversionofavidin,knownasNeutrAvidinProtein,withamassofapproximate-ly60,000daltons.Asaresultofcarbohydrateremoval,lectinbind-ingisreducedtoundetectablelevels,yetbiotin-bindingaffinityisretainedbecausethecarbohydrateisnotnecessaryforthisactiv-ity.NeutrAvidinProteinofferstheadvantagesofanear-neutralpI(6.3)tominimizenonspecificadsorption,alongwithlysineresiduesthatremainavailableforderivatizationorconjugation.NeutrAvidinProteinyieldsthelowestnonspecificbindingamongtheknownbiotin-bindingproteinsduetoitsnear-neutralpIandlackofbothcarbohydrateandRYDsequence.
Avidin:Biotin Binding
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 67
A Comparison of Biotin-Binding Proteins
Thestrongassociationbetweenavidinandbiotincanbeusedinthefieldofaffinityseparations.Byattachingavidintoasolidsupport,abiotinylatedproductcanbeanchoredtothesamesolidsupport.TheattachmentisstableoverawiderangeofpH,saltconcentrationsandtemperatures.Todissociatebiotinfromavidin,8Mguanidine•HCl,pH1.5orboilinginSDS-PAGEsamplebuffermustbeused.
Avidin
Streptavidin
Thermo Scientific NeutrAvidin Protein
Molecular Weight 67K 53K 60K
Biotin-binding Sites 4 4 4
Isoelectric Point (pl) 10 6.8–7.5 6.3
Specificity Low High Highest
Affinity for Biotin (Kd) 10-15M 10-15M 10-15M
Nonspecific Binding High Low Lowest
Immobilized Avidin Products
Strong biotin interaction creates a nearly irreversible bond.
Immobilizedavidincanbeusedinavarietyofapplicationsfortheaffinitypurificationofbiotinylatedmacromolecules.Inonevariation,anantibodythathasanaffinityforaparticularantigenislabeledwithbiotin.Cellscontainingtheantigenarelysed,thenincubatedwiththebiotinylatedantibodytoformatypicalantigen/antibodycomplex.Toisolatetheantigen,thecrudemixtureispassedthroughanimmobilizedavidinorstreptavidincolumn,whichwillbindthecomplex.Afterappropriatewashes,theantigencanbeelutedfromthecolumnwithalowpHelutionbuffer.Thebiotinylatedantibodyisretainedbythecolumn.
Applications:•Bindingbiotinylatedanti-transferrinforpurifyingtransferrin fromserum1
•BindingbiotinylatedpeptidesandelutionwithanSDS/ureasolution2
•HybridizationofbiotinylatedRNAtoitscomplementaryDNA andbindingtoimmobilizedavidin,withsubsequentelutionofthe single-strandedDNA3
•Purificationofdouble-strandedDNA4
References1.Wilchek,M.andBayer,E.A.(1989).Protein Recognition of Immobilized Ligands.
Hutchins,T.W.,ed.AlanR.Liss,Inc.,pp.83–90.2.Swack,J.A.,et al.(1978).Anal. Biochem.87,114–126.3.Manning,J.,et al.(1977).Biochemistry16, 1364–1370.4.Pellegrini,M.,et al.(1977).Nucleic Acids Res.4,2961–2973.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20219 Avidin Agarose Resin Support:Crosslinked6%beadedagaroseCapacity:≥20μgbiotin/mLresin
5mL $150
20225 Avidin Agarose Resin SupportandCapacity:Sameasabove
5x5mL $533
20362 Avidin Agarose ColumnsSupportandCapacity:Sameasabove
5x1mL $190
Immobilized Streptavidin Products
Same high biotin-binding affinity as avidin with low nonspecific binding.
Applications:•Purificationofmembraneantigensinconjunctionwith biotinylatedmonoclonalantibodies1,2
•Cell-surfacelabelingwithbiotinylationreagents,followedby precipitationwithimmobilizedstreptavidin3
•Purificationofcell-surfaceglycoproteinsusingbiotinylated ConcanavalinA4
•Recoveryofsingle-strandedDNAfordideoxysequencing5
References1.Gretch,D.R.,et al.(1987).Anal. Biochem.163,270–277.2.Updyke,T.V.andNicolson,G.L.(1984).J. Immunol. Method73,83–95.3.Lisanti,M.P.,et al.(1989).J. Cell Biol.109,2117–2127.4.Buckie,J.W.andCook,G.M.(1986).Anal. Biochem.156(2),463–472.5.Baqui,M.,et al.(2003).J. Biol. Chem.278,1206–1211.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20347 Streptavidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:1–3mgbiotinylatedBSA/mLresin 15–28μgbiotin/mLresin
2mL $178
20349 Streptavidin Agarose ResinSupportandCapacity:Sameasabove
5mL $310
20353 Streptavidin Agarose Resin SupportandCapacity:Sameasabove
10mL $440
20351 Streptavidin Agarose ColumnsSupportandCapacity:Sameasabove
5x1mL $318
53113 Streptavidin UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥2mgbiotinylatedBSA/mLresin ≥24μgbiotin/mLresin
2mL $212
53114 Streptavidin UltraLink ResinSupportandCapacity:Sameasabove
5mL $370
53116 Streptavidin Plus UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥4mgbiotinylatedBSA/mLresin ≥48μgbiotin/mLresin
2mL $285
53117 Streptavidin Plus UltraLink ResinSupportandCapacity:Sameasabove
5mL $410
20357 High Capacity Streptavidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:>10mgbiotinylatedBSA/mLofresin
2mL $225
20359 High Capacity Streptavidin Agarose ResinSupportandCapacity:Sameasabove
5mL $350
20361 High Capacity Streptavidin Agarose ResinSupportandCapacity:Sameasabove
10mL $600
21344 MagnaBind Streptavidin BeadsSupport:1–4μm,ironoxideparticlesCapacity:2μgbiotin/mLbeads
5mL $271
88816 Pierce Streptavidin Magnetic Beads 1mL $213
88817 Pierce Streptavidin Magnetic Beads 5mL $721
68 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Immobilized NeutrAvidin Products
Less nonspecific binding produces cleaner results and better yields.
Whennonspecificbindingisaprobleminyourapplication,ThermoScientificImmobilizedNeutrAvidinProductsaresuperioralternativestoavidinorstreptavidin.NeutrAvidinBiotin-BindingProteinisamodifiedavidinderivativethatcombinesseveralkeyfeaturestoprovidebiotin-bindingwithexceptionallylownonspecificbindingproperties.
Highlights:•Carbohydrate-free–justlikestreptavidin,NeutrAvidinBiotin- BindingProteinhasnocarbohydrate,eliminatingnonspecific bindingproblemsduetosugars•Nointeractionwithcellsurfacemolecules–absenceofthe Arg-Tyr-Aspsequence(presentinstreptavidin),whichmimics theuniversalcellsurfacerecognitionsequencepresentina varietyofmolecules,eliminatescross-reactivityofcellsurface molecules•Neutralpl–withaplof6.3,NeutrAvidinProteinhasaplthatis closertoneutralitythanavidinorstreptavidin,eliminating electrostaticinteractionthatcontributestononspecificbinding
Applications:•Immunoprecipitation•Purifyingproteinsthatbindtobiotinylatedligands•Capturingbiotinylatedcell-surfaceproteins1-3
•Purifyingbiotinylatedpeptides4
Ordering Information Product #
Description
Pkg. Size
U.S. Price
29200 NeutrAvidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:>20μgor80nmolbiotin/mLresin (approx.1–2mgbiotinylatedBSA/mLresin)
5mL $205
29201 NeutrAvidin Agarose Resin SupportandCapacity:Sameasabove
10mL $350
53150 NeutrAvidin UltraLink ResinSupport:UltraLinkBiosupportCapacity:12–20μgbiotin/mLgel
5mL $255
53151 NeutrAvidin Plus UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥30μgbiotin/mLgel
5mL $340
29202 High Capacity NeutrAvidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:>75μgbiotin/mLresin >8mgbiotinylatedBSA/mLresin
5mL $250
29204 High Capacity NeutrAvidin Agarose ResinSupportandCapacity:Sameasabove
10mL $410
Immobilized Monomeric Avidin and Kit
Ideal affinity support for gentle, reversible binding of biotinylated proteins.
Tobreaktheavidin-biotininteraction,8Mguanidine•HClatpH1.5orboilinginSDS-PAGEsamplebufferisrequired.Theseelutionmethodsmayresultindenaturationofthebiotinylatedproteinandcauseirreversibledamagetothesupport.Inaddition,avidinorstreptavidinwillbeirreversiblydenaturedandlosetheabilitytobindsubsequentbiotinylatedsamples.
Whenavidiniscoupledtoasolidsupportasthesubunitmonomer,thespecificityforbiotinisretained,buttheaffinityforbiotinbind-ingsubstantiallydecreases(Ka~108M-1).TheMonomericAvidinAgaroseResinandKitcanbeusedtobindbiotinylatedmolecules,andtheboundmaterialcanbecompetitivelyelutedusing2mmbiotininphosphate-bufferedsaline(PBS).Thistechniqueprovidesthegentlestelutionconditionswithoutcontaminationoftheavidinsubunitsorsubstantiallossofcolumn-bindingcapacity.
Highlights:•Purifiesbiotinylatedproductsundermildelutionconditions•Canberegeneratedandreusedatleast10times•Exhibitslittlenonspecificbinding(3%orless)
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20228 Monomeric Avidin Agarose Resin Support:Crosslinked4%beadedagaroseCapacity:≥1.2mgbiotinylatedBSA/mLresin
5mL $355
20267 Monomeric Avidin Agarose ResinSupportandCapacity:Sameasabove
10mL $570
20227 Monomeric Avidin Agarose KitSupportandCapacity:SameasaboveIncludes:1x2mLColumn,BindingandElutionbuffers
Kit $341
53146 Immobilized Monomeric Avidin UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥1.2mgbiotinylatedBSA/mLresin
5mL $399
29129 Biotin 1g $ 54
Thermo Scientific Products for Avidin:Biotin Binding
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 69
Immobilized Iminobiotin and Biotin
Iminobiotin offers mild dissociation conditions at pH 4.
O
NHHN
S
NH
Immobilized Iminobiotin
HN
HN
HN
Aga
rose
Bea
d
Iminobiotinistheguanidoanalogofbiotin.Thedissociationconstantoftheavidin-iminobiotincomplexispH-dependent.AtpH9.5-11.0,theavidin-iminobiotincomplexwillbindtightly.AtpH4,theavidin-iminobiotincomplexwilldissociate.Becausedenaturingagentssuchas8Mguanidine•HCIor4Mureaarenotusedinthepurification,anavidinconjugatehasabetterchanceofmaintainingitsactivityduringpurification.
UseimmobilizedD-Biotinasan“irreversiblelinkage”tobindstreptavidinconjugates.Thebiotin-streptavidininteractioncanwithstandextremesinpH,saltanddetergents.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20221 Iminobiotin Agarose ResinSupport:Crosslinked6%beadedagaroseSpacer:DiaminodipropylamineCapacity:≥1mgofavidin/mLresin
5mL $119
20218 Biotin Agarose ResinSupport:PierceCDISupportSpacer:DiaminodipropylamineCapacity:≥2mgofavidin/mLresin
5mL $ 99
70 For more information, or to download product instructions, visit www.thermoscientific.com/pierce
Thermo Scientific Pierce Chromatography Cartridges are convenient, reliable, ready-to-use, pre-packed 1mL and 5mL columns of sample-prep and affinity purification resins for manual or automated liquid chromatography (LC).
Thecartridgefittingsarecompatiblewiththepopularautomatedliquid-chromatographysystemsorformanualsyringeprocessing.ThecartridgesattachdirectlytoÄKTAorFPLCSystemswithoutadditionalconnectors.Cartridgescanbeusedindividuallyorconnectedinaseriestoobtainevenhighercolumncapacity.EachproductsuppliedinthePierceChromatographyCartridgeformataccessorypackthatreadilyadaptscartridgesforuseusingLuer-LokSyringeFittingsortubing.Thecartridgesprovidefast,easyandreproduciblechromatographicseparationsandcanberegeneratedformultipleuses.
Thermo Scientific FPLC Cartridges shematic.
Chromatography Cartridge Highlights• Two sizes–1mLand5mL,convenientfortypicalresearchscales• Compatible–fittingsallowconnectionwithpopularLCsystems
orastandardsyringe• Versatile–usesinglyorconnectedinseriestoservicedifferent
capacityrequirements• Validated–availablecartridgeshavebeentestedtoensure
performanceintheformat• Reusable–accessorypackincludescapsforconvenient
storagebetweenuses• Economical–comparableperformanceatlowercostthanother
commerciallyavailablecartridges
Thermo Scientific Pierce Chromatography Cartridge properties.
1mL Cartridge 5mL Cartridge
Dimensions 0.7x2.7cm 1.3x3.8cm
RecommendedFlowRate 1-4mL/min 1-7mL/min
MaximumPressure 0.3mPa(43psior3bar) 0.3mPa(43psior3bar)
CartridgeMaterial polypropylene polypropylene
FritMaterial polyethylene polyethylene
Applications for Pierce Chromatography Cartridges:•His-taggedproteinpurification•GST-taggedproteinpurification•Antibodypurification•Biotinbinding•Phosphoproteinenrichment•Proteindesalting
FPLC Cartridge Overview
20mm
Port Diameter: 4.2mm10-32 threaded coned port
14mm
4mm
17.5
mm
17.5
mm
89m
m 13.5mm
Port Diameter: 4.2mm10-32 threaded coned port
8.5mm
4mm
12.5
mm
75m
m
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 71
His-tagged Protein FPLC Purification
Thermo Scientific HisPur Nickel-NTA CartridgesDelivers the highest yield of His-tagged protein with a re-useable resin.
Highlights:•Bindupto60mgof6xHis-taggedproteinpermilliliterofresin•Purifyproteinsusingnativeordenaturingconditions•UsewithPierceCellLysisReagentsandavarietyofbufferadditives•Reusecartridgesseveraltimes
0 20 40 60 80 100 120 140mL0
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Purification of 6xHis-GFP from E. coli lysate using a Thermo Scientific HisPur Nickel-NTA Cartridge. Bacteriallysate(130mgtotalprotein)containingover-expressed6xHis-GFP(greenfluorescentprotein)wasdiluted1:1withequilibra-tionbufferandappliedtoaHisPurNi-NTAChromatographyCartridgeataflowrateof1mL/min.ThecartridgewaswashedwithPBS,68mMimidazoleuntilthebaselineabsorbancewasreached.The6xHis-GFPwaselutedwithPBS,300mMimidazole.Left panel:6xHis-GFPelutionwasmonitoredat280nm(blackline)and485nm(redline;GFP-specific).Right panel:SelectedfractionswereanalyzedbySDS-PAGE.Gellaneswerenormalizedtoequivalentvolume.M =MWmarker,L =lysateload,FT =flow-throughandE =elution.
Seepage14formoreinformationandotherpackagingformatsofHisPurNi-NTAResin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
90098 HisPur Ni-NTA Chromatography Cartridges, 1mL Formulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding up to 60mg of His-tagged protein per cartridge.
5cartridges $155
90099 HisPur Ni-NTA Chromatography Cartridges, 5mL Formulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding up to 300mg of His-tagged protein per cartridge.
2cartridges $200
Thermo Scientific HisPur Cobalt CartridgesHighly selective binding for the highest purity His-tagged protein purification.
Highlights:•Obtainmorethan10mgofpureHis-taggedproteinpermilliliter
ofresinwithoutoptimizingimidazolewashingconditions•Cobalt-chelatecoordinationcorebindsfewerhostproteincon-
taminants,resultinginlowerbackgroundthannickelresins•Nometalcontaminationinelutedhistidine-taggedproteinsample•Purifyproteinsundernativeordenaturingconditions;compatible
withPierceCellLysisReagentsandavarietyofbufferadditives•Reusecartridgesseveraltimes
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Purification of 6xHis-GFP from E. coli lysate using a Thermo Scientific HisPur Cobalt Cartridge. His-taggedgreenfluorescentprotein(GFP)wasextractedfromE. coliusingThermoScientificB-PERBacterialProteinExtractionReagentinPhosphateBuffer(Product#78266)containingThermoScientificHaltProteaseInhibitorCocktail,EDTA-Free(Product#78415).Thelysatewasdiluted1:1withequilibration/washbuffer(50mMsodiumphosphate,300mMsodiumchloride,10mMimidazole,pH7.4)andappliedtoaHisPurCobaltChromatographyCartridgeataflowrateof0.3mL/min.Thecartridgewaswashedwithequilibration/washbufferuntilthebaselineabsorbanceat280nmwasreached.His-taggedGFPwaseluted(50mMsodiumphosphate,300mMsodiumchloride,150mMimidazole;pH7.4)andselectedfractionswereanalyzedbySDS-PAGEandThermoScientificGelCodeBlueStainReagent(Product#24592).M =MWMarker;S=non-fractionatedlysate;FT =flow-through.
Seepage16formoreinformationandotherpackagingformatsofHisPurNi-CobaltResin.
72 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Ordering Information Product #
Description
Pkg. Size
U.S. Price
90093 HisPur Cobalt Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 10mg of His-tagged protein per cartridge.
5cartridges $160
90094 HisPur Cobalt Chromatography Cartridges, 5mLFormulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 50mg of His-tagged protein per cartridge.
2cartridges $267
GST-Tagged Protein FPLC Purification
Thermo Scientific Pierce Glutathione Agarose CartridgesCost-effective and high-performance GSH resins for purification of recombinant GST fusion proteins.
Highlights:•Bindsatleast25mgofrecombinantGSTproteinpermilliliter
ofresin•Consistentlypurifiesatleast10mgofGST-taggedproteinper
milliliterofresinwithgreaterthan90%purity•Economicallypricedandcanbereusedatseveraltimeswithout
reductioninbindingcapacityandpurificationperformance•WorkswelltopurifyGST-fusionproteinsfrombacteriallysates
orusewithpre-purifiedGST-taggedproteinstopulldownproteininteractions
•ValidatedandeffectiveforusewithPierceCellLysisReagentstoextractandpurifyfrombacterialormammaliancellcultures
Seepage18formoreinformationandotherpackagingformatsofPierceImmobilizedGlutathioneAgaroseResin.
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Wash Elutionof
11.8 mg pureGST-fusion protein
0.0 10.0 20.0 30.0 40.0 50.0 60.0mL
Purification of Pak 1-GST fusion protein on Thermo Scientific Pierce Glutathione Chromatography Cartridge 1mL.
Sample:50mg(10mL)clarifiedE. colilysatecontainingexpressedGST-Pak1,M34,000BindingBuffer:5mMTris,150mMsodiumchloride,pH8.0ElutionBuffer:50mMTris,150mMsodiumchloride,10mMGlutathione,pH8.0FlowRate:Loadat0.5mL/min,Wash,Elute1mL/minInstrument:AKTApurifier
16.5
47
3225
11080
210
M Load FT Wash Elution
Purification of Pak 1-GST fusion protein using the Pierce GST Spin Purification Kit (Product # 16106). Pak1-GSTlysate(2.4mgtotalprotein)wasappliedinGlutathioneBindingBuffertoa0.2mLPierceGlutathioneSpinColumnandelutedwith10mMGlutathioneElutionBuffer,pH8.0.FractionswereresolvedbySDS-PAGEusinga4-20%Tris-Glycinegel.GelwasstainedwithGelCodeBlueStainReagent(Product#24590).Pierce3-ColorProteinMolecularWeightMarkerMix(Product#26691)wasused.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
16109 Pierce Glutathione Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding approx. 10mg of GST-tagged protein per cartridge.
5cartridges $230
16110 Pierce Glutathione Chromatography Cartridges, 5mLFormulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding approx. 50mg of GST-tagged protein per cartridge.
2cartridges $400
FPLC Cartridges
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 73
Antibody FPLC Purification
Thermo Scientific Pierce Protein A Agarose CartridgesIdeal for purification of IgG from serum and other fluids.
Highlights:•Bindstoawiderangeofantibodies–especiallygoodfor
purificationofrabbitIgG•LessexpensivethanProteinGagarose
M L FT EFT
FT E
E
A28
0 A
280
M L FT E
50.0mL40.030.020.010.00.0
50.0mL40.030.020.010.00
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0.0
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Comparable antibody yield and purity achieved with Thermo Scientific Pierce Protein A Chromatography Cartridge. Normalhumanserum(60mg)wasappliedinPBStoa1mLThermoScientificPierceProteinACartridge(top)andaHiTrap®Column(bottom)andelutedwith0.1Mglycine,pH2.8,usingaflowrateof1mL/minute.Thearrowdenotesthestartofthelow-pHelution.TheyieldofhumanIgGwas6.85mgand6.88mg,respectively.FractionswereseparatedbySDS-PAGEandthegelswerestainedwithThermoScientificImperialProteinStain(Product#24615).M =MWmarker,L =sampleload,FT =flow-throughandE =elution.
Seepage60formoreinformationandotherpackagingformatsofPierceProteinAAgaroseResin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89924 Pierce Protein A Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding 35mg human IgG per cartridge.
2cartridges $170
89925 Pierce Protein A Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-Lokfittings.Sufficient for: Binding 175mg human IgG per cartridge.
1cartridge $335
Thermo Scientific Pierce Protein G Agarose CartridgesEspecially suited for purification of monoclonal antibodies from mouse and the broadest spectrum of species and IgG subclasses from human, goat and sheep samples.
Highlights:•Albuminandcellsurfacebindingsitehavebeenremovedfrom
ProteinGtoprovidehigherantibodypurity•BindstoawiderrangeofantibodiesthanProteinAbeads
M L FT ElutionFT
FT E
E
A28
0A
280
M L FT Elution
35.0mL30.020.0 25.010.0 15.05.00.0
35.0mL30.020.0 25.010.0 15.05.00.0
0
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0
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Comparable antibody yield and purity acheived with Thermo Scientific Pierce Protein G Chromatography Cartridge. Normalhumanserum(60mg)wasappliedinPBStoa1mLThermoScientificPierceProteinGCartridge(top)anda1mLHiTrapColumn(bottom)andelutedwith0.1Mglycine,pH2.8,usingaflowrateof1mL/minute.ThearrowdenotesthestartofthelowpHelution.FractionswereseparatedbySDS-PAGEandthegelswerestainedwithImperial®ProteinStain(Product#24615).M =MWmarker,L =sampleload,FT =flow-throughandElution=elutionfractions.
Seepage60formoreinformationandotherpackagingformatsofPierceProteinGAgaroseResin
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89926 Pierce Protein G Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-LokFittings.Sufficient for: Binding 11 to 15mg human IgG per cartridge.
2cartridges $190
89927 Pierce Protein G Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: Binding 55 to 75mg human IgG per cartridge.
1cartridge $390
74 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Thermo Scientific Pierce Protein A/G Agarose CartridgesGenetically-engineered protein that combines the IgG binding domains of both Protein A and Protein G.
Highlights:•SinglesupportprovidesallbenefitsofProteinAandProteinG•BindsawiderrangeofantibodiesthanProteinAand
ProteinGbeads
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A)
PureRabbitIgG
B)
M S FT E
RabbitIgG
kDa210
110
80 47
32 25
A28
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Thermo Scientific Pierce Protein A/G Chromatography Cartridges are effec-tive for affinity purification of immunoglobins from serum. A) 2mLofrabbitserumwasappliedtothecartridgeandtheresultingchromatogramrecorded.B) FractionswereanalyzedbySDS-PAGEona4-20%TrisGlycinegelstainedwithImperialProteinStain(Product#24615).M=Markerproteins,S=Sampleapplied,FT=Flow-throughduringsampleloadandwash,E=ElutedrabbitIgG.ThearrowindicatesthelocationoftheisolatedIgG.
Seepage61formoreinformationandotherpackagingformatsofPierceProteinA/GAgaroseResin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89930 Pierce Protein A/G Chromatography Cartridges, 1mL Formulation:1mLresincartridgeswithLCandLuer-LokFittings.Sufficient for: Binding 7mg human IgG per cartridge.
2cartridges $200
89931 Pierce Protein A/G Chromatography Cartridges, 5mL Formulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: Binding 35mg human IgG per cartridge.
1cartridge $399
Thermo Scientific Pierce Protein L Agarose CartridgesGreat for purifying ScFv or Fab fragments and monoclonal antibodies containing kappa light chains.
Highlights:•Bindskappalightchainsfromawiderangeofspecieswithout
interferingwithantigen-bindingsites†
•BindstoallclassesofIg(e.g.,IgG,IgM,IgA,IgEandIgD)†
•Bindssingle-chainvariablefragments(ScFv)†
•Doesnotbindbovine,goatorsheepIgs•Doesnotbindtobovineantibodies,makingitidealforpurification
ofmouseIgG†fromcellculturesupplementedwithbovineserum† Note: Lambda light chains and some kappa light chains will not bind. Binding will only occur if the appropriate kappa light chains are present.
0 50100150200250300350
mAU
0.0 10.0 20.0 30.0mLA1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A12 B11 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4
M L Flow-through Wash Eluate
SampleApplication
kDa210
80
47
32 25
16.5
IgA
A)
A28
0
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IgAheavychain
kappa lightchain
110
Thermo Scientific Pierce Protein L Chromatography Cartridges isolate and purify immunoglobulin classes IgG, IgM, IgA, IgE and IgD via their kappa light chains.A) HumanIgASerum(2mg)wasappliedin100mMsodiumphosphate,150mMsodiumchloride,pH7.2toa1mLPierceProteinLChromatographyCartridgeandpurifiedataflowrateof1mL/minute.Targetwaselutedin0.1Mglycine,pH2.8.B) FractionswereanalyzedbySDS-PAGEona4-20%Tris-GlycinegelstainedwithGelCodeBlueStainReagent(Product#24590).
Seepage61formoreinformationandotherpackagingformatsofPierceProteinLAgaroseResin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89928 Pierce Protein L Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding 5 to 10mg human IgG per cartridge.
2cartridges $190
89929 Pierce Protein L Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: Binding 25 to 50mg human IgG per cartridge.
1cartridge $375
FPLC Cartridges
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 75
Thermo Scientific Melon Gel IgG Purification ResinsEasily purify IgG from serum in 15 minutes.
Highlights:•Notediousbinding,washingandmultipleelutionsteps•Purifiesantibodiesfromserumfourtosixtimesfasterthan
ProteinAorGmethods•Recoverantibodiesfrommanyspecieswith>80%purity•Noharshelutionconditionsmeansantibodiesretainmoreactivity•Reusablesupport
M L
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A28
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Effective reverse-affinity purfication of antibody with Thermo Scientific Melon Gel Cartridges. Normalhumanserum(1mL)wasappliedtoa1mLMelonGelCartridgeataflowrateof1mL/minute.Proteincontaminantsbindtotheresinwhiletheantibodiesflowthrough(FT).Volumeofrecoveredantibody=4to5mL.Regeneration(R)solutionstripstheboundproteincontaminantssothatthecartridgecanbereusedmultipletimes.ThepurityoftheisolatedantibodywasevaluatedbySDS-PAGE(rightpanel)andstainedwithThermoScientificGelCodeBlueStainReagent(Product#24590).M =MWMarker,L =sampleloaded,FT =flow-throughandR =regenerationfractions.
Seepage63formoreinformationandotherpackagingformatsofMelonGelResin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89932 Melon Gel Chromatography Cartridges, 1mL Formulation:1mLresincartridgeswithLCandLuer-LokFittings.Sufficient for: IgG purification from 1 to 2mL serum per cartridge.
2cartridges $100
89933 Melon Gel Chromatography Cartridges, 5mL Formulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: IgG purification from 5 to 10mL serum per cartridge.
1cartridge $201
Phosphoprotein FPLC Purification
Thermo Scientific Pierce Phosphoprotein Enrichment KitPurify and isolate phosphoproteins for analysis by Western blotting or mass spectrometry.
Highlights:•Lownonspecificproteincontaminationfromcomplexbiological
samples,suchascellculturelysateandmousetissueextract•Easyspinformatenablesenrichmentofphosphorylatedproteins
inlessthan2hours•Achieveshigheryieldsthanothercommerciallyavailablekits
Phosho-p42/44 MAPKCytochrome-C (12KD)
Non-phosphorylated protein
25ug 10ug FT El25ug 10ug FT El TCL TCL
A)
B)
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10.00.0 20.0 30.0 40.0 50.0 60.0 70.0mL
Non-phosphorylatedProteinsFlow-Through
ConcentratedPhosphorylatedProteins In Eluate
A28
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Figure A. Enrichment of phosphorylated proteins from K562 cell lysate, 4mg (10mL), processed using a Thermo Scientific Pierce Phosphoprotein Enrichment Chromatography Cartridge, 1mL.Binding/WashBuffer:50mMMES2-(N-morpholino)ethanesulfonicacid,monohydrate),250mMsodiumchloride,25mMadipicacid,0.25%CHAPS;pH5.0;ElutionBuffer:100mMsodiumphosphate,500mMsodiumchloride,0.25%CHAPS;pH7.5;Flowrateduringsampleapplication0.3mL/min;1mL/minduringwashandelution.Figure B. Thermo Scientific Pierce Phosphoprotein Enrichment Chromatography Cartridge, 1mL, provides high specificity for the enrichment of phosphoproteins from complex biological samples. ConcentratedFlow-throughandElutionfractionswereresolvedonSDS-PAGE.Gellaneswerenormalizedbyproteinconcentration,10mg/lane.Westernblotanalysiswasperformedusingphospho-specificantibodies.CytochromeCisanegativecontrolfornonspecificbindingofnon-phosphorylatedproteins.Highspecific-ityofbindingaffinityforthephosphorylatedtargetwasdemonstratedbythepresenceofPhospho-p42/44MAPKintheEluateandabsence,thereof,intheFlow-through.
Seepage53formoreinformationandotherpackagingformatsofPierceProteinPhosphoproteinEnrichmentResin.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
87743 Pierce Phosphoprotein Enrichment Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Purifying samples containing 4mg total protein (400µg phosphoprotein) per cartridge.
2cartridges $124
87744 Pierce Phosphoprotein Enrichment Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-Lokfittings.Sufficient for: Purifying samples containing 20mg total protein (2mg phosphoprotein) per cartridge.
1cartridge $310
76 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Biotinylated Protein FPLC Purification
Thermo Scientific Streptavidin Agarose and Neutravidin Agarose CartridgesResins with high biotin affinity and low nonspecific binding.
Highlights:•ExhibithighbindingaffinitytowardsthevitaminBiotin(vitaminH)•Capturesbiotinylatedproteins•Derivativesoftheproteinavidinfromchickeneggwhitesthat
differbytheirsourceandglycosylationlevel
Pierce High Capacity Streptavidin Chromatography Cartridge
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500
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3500
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Yield655 µgTarget
GE HiTrap Streptavidin HP
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500
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Yield469 µgTarget
Pierce High Capacity NeutrAvidin Chromatography Cartridge
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3000
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Yield388 µgTarget
Thermo Scientific High Capacity Streptavidin Chromatography Cartridge achieves comparable yield and performance in binding of small biotinylated molecule, Biotin 4 Nitrophenyl ester (BpNPE), as compared to HiTrap.
Sample:4.8mgBpNPE(in24mL)ColumnSize:1mLBindingBuffer:0.5MSodiumAcetate,pH5.0ElutionBuffer:0.5MNaOHStreptavidincartridges;0.1MNaOHNeutrAvidincartridgeFlowRate:SampleApplication0.3mL/minute;WashandElution1mL/minuteMonitored2wavelengths:Flowthrough/WashinNaAcetateA270nm(red);ElutioninNaOHA410nm(pink)Instrument:AKTApurifierYielddeterminedbyODA410nm(ExtinctionCoefficientforBpNPE18.3A410nm)
SupplierCartridge
SizeBiotinylated BSA Bound
Pierce High Capacity Streptavidin Chromatography Cartridge
1mL 12.9mg
5mL 75.9mg
GE HiTrap Streptavidin HP
1mL 10.7mg
5mL (Notofferedin5mLsize)
Pierce High Capacity NeutrAvidin Chromatography Cartridge
1mL 12.8mg
5mL 70.mg
Binding capacity of Thermo Scientific High Capacity Streptavidin Chromatography Cartridges is comparable to that of HiTrap. ColumnswereoverloadedwithBiotinylatedBSAandpurifiedpermanufacturer'sinstructions.BindingcapacitywasdeterminedusingtheThermoScientificPierceBCAProteinAssayKit(Product#23225).Note: Capacity for the avidin resins was determined indirectly by subtracting the unbound biotinylated BSA present in the flow-through fractions from the total amount applied to the column.
Comparison of biotin-binding proteins.
Biotin-Binding Protein
Avidin Streptavidin NeutrAvidin
Molecular Weight 67K 53K 60K
Biotin-binding Sites 4 4 4
Isoelectric Point (pI) 10 6.8-7.5 6.3
Specificity Low High Highest
Affinity for Biotin (Kd) 10-15M 10-15M 10-15M
Nonspecific Binding High Low Lowest
Seepages67-68formoreinformationandotherpackagingformatsofSteptavidinandNeutravidinAgaroseResin
Ordering Information Product #
Description
Pkg. Size
U.S. Price
87739 High Capacity Streptavidin Chromatography Cartridges, 1mL Formulation:1mLcrosslinked6%beadedagarosecartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 100µg biotin per cartridge (> 10mg biotinylated BSA per cartridge).
2cartridges $176
87740 High Capacity Streptavidin Chromatography Cartridge, 5mL Formulation:5mLcrosslinked6%beadedagarosecartridgewithLCandLuer-Lokfittings.Sufficient for: Binding > 500µg biotin per cartridge (> 50mg biotinylated BSA per cartridge).
1cartridge $240
87741 High Capacity NeutrAvidin Chromatography Cartridges, 1mL Formulation:1mLcrosslinked6%beadedagarosecartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 75µg biotin per cartridge (> 8mg biotinylated BSA per cartridge).
2cartridges $149
87742 High Capacity NeutrAvidin Chromatography Cartridge, 5mL Formulation:5mLcrosslinked6%beadedagarosecartridgewithLCandLuer-Lokfittings.Sufficient for: Binding > 375µg biotin per cartridge (> 40mg biotinylated BSA per cartridge).
1cartridge $206
FPLC Cartridges
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 77
Protein Desalting
Thermo Scientific Zeba Desalting Chromatography CartridgesThe best protein desalting resin in cartridge format.
Highlights:•Exceptionaldesaltingandprotein-recoverycharacteristics
comparedtoothercommerciallyavailableresins•Successfulwithverydilute(25μg/mL)proteinsamples•Greaterthan95%retention(removal)ofsaltsandother
smallmoleculesandgoodrecoveryofproteinsandothermacromolecules
A28
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Thermo Scientific Pierce Chromatography Cartridge
Supplier G Chromatography Cartridge
Supplier B Chromatography Cartridge
mAU
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100
0
0.0 2.0 4.0 6.0 8.0 10.0mL
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Efficient salt removal and protein recovery with desalting chromatography cartridge. Bovineserumalbumin(1mg)in1MNaClwasappliedto5mLThermoScientificZebaDesaltingCartridge(top)ataflowrateof5mL/minute.CartridgeprofileshowsisocraticelutionofBSA(blue)andNaCldetectedbyconductivity(brown).Greaterthan95%oftheBSAwasrecoveredandmorethan95%ofthewassaltremoved.Resultswerecomparabletothoseobtainedwithdesalt-ingcartridgesfromothersuppliers(resultsfortheThermoScientificCartridgewereessentiallyidenticaltothatobtainedwithmoreexpensiveGEHealthcareandBio-RadProducts(suppliersGandB,respectively).
Ordering Information Product #
Description
Pkg. Size
U.S. Price
89934 Zeba Desalting Chromatography Cartridges, 7K MWCO, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Desalting 50 to 250µL samples per use.
5cartridges $147
89935 Zeba Desalting Chromatography Cartridges, 7K MWCO, 5mLFormulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Desalting 100 to 1500µL samples per use.
5cartridges $168
78 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
In addition to the few affinity supports whose ligands have broad application to many different protein methods, there are many others whose applications are more narrowly defined or are incorporated into kits for very specific purposes. TheseincludekitstoisolatecellsurfaceproteinsusingbiotinylationandNeutrAvidinAgarose,kitstoenrichforphosphoproteins,glyco-proteinsandubiquitinatedproteins.Thestand-aloneresinsonthispageandthenextincludethosethatcanbeusedtoremovetrypsinfromproteindigest,isolatebloodproteins,purifyC-reactiveproteinandcaptureribonucleosides.Inaddition,page80presentsourcompletelineofmagneticbeads,togetherwiththeirmagnetaccessories.
Immobilized Soybean Trypsin Inhibitor
For effective removal of trypsin, chymotrypsin and elastase from protein digests.
Applications:•Purifyingtrypsin,chymotrypsinandelastase1,2
•Removingproteasesfromactivatedpancreaticjuices3
References1.Feinstein,G.,et al.(1974).Euro. J. Biochem.43(3),569–581.2.Peterson,L.M.,et al.(1976).Biochemistry15(12),2501–2508.3.Reeck,G.R.,et al.(1971).Biochemistry10(25),4690–4698.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20235 Immobilized Soybean Trypsin InhibitorSupport: 4%beadedagaroseCapacity:≥6mgtrypsin/mLofresin
2mL $98
Immobilized Pepstatin
An excellent cathepsin binding matrix.
HN
HN
HN
Pepstatin =
R = (4-amino-3-hydroxy-6-methyl) heptanoic acid
Thermo Scientific Immobilized Pepstatin
i-ValValValRAlaR
Pepstatin
Aga
rose
Bea
d
Immobilized Heparin
ReferenceHelseth,Jr.,D.L.andVeis,A.(1984).Proc. Natl. Acad. Sci. USA81,3302–3306.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20215 Immobilized PepstatinSupport:Crosslinked6%beadedagaroseSpacer:DiaminodipropylamineCapacity:1–2mgofpepsin/mLofresin
5mL $159
Additional Thermo Scientific Affinity Supports and Kits
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 79
Immobilized Heparin
Use to isolate many blood proteins that have enzymatic activities.
Applications:•Enrichlysatesfornucleicacid-bindingproteins•Isolatemanybloodproteins
Aga
rose
Bea
d
O
OSO3-
OHCOO-
O
NHOSO3-
OH
CH2OSO3-
OO
n
O
Thermo Scientific Immobilized Heparin
ReferenceSmith,P.K.,et al.(1980).Anal. Biochem.109,466–473.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20207 Immobilized HeparinSupport:4%beadedagaroseLoading:≥0.2mgofheparin/mLofresin(determinedbythecolorimetricmethod)
Kit $73
Immobilized p-Aminophenyl Phosphoryl Choline
For C-reactive protein binding.
O
ON+P
O
O
O-
HN
Thermo Scientific Immobilized p-Aminophenyl Phosphoryl Choline
Aga
rose
Bea
d
ReferenceRobey,F.A.andLiu,T.Y.(1981).J. Biol. Chem.256,969–975.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20307 Immobilized p-Aminophenyl Phosphoryl CholineSupport:Crosslinked6%beadedagaroseCapacity:≥3mgofhumanC-reactiveprotein/mLofresin
5mL $202
Immobilized D-Galactose
For lectin and galactosidase binding.
Applications:•Humanalpha-galactosidaseApurification1
•E. coliheatlabileenterotoxinpurification2
•C-typelectinpurification3
•Choleratoxin(CT)purification4,5
O
S
O
O
O OH
OHOHO
OH
Thermo Scientific Immobilized Thio-alpha-D-Galactose
Aga
rose
Bea
d
References1.Yasuda,K.,et al.(2004).Protein Expression and Purification.37,499–506.2.Okamoto,K.,et al.(1998).J. Bacteriol.180,1368–1374.3.Matsumoto,J.,et al.(2001).Development. 128,3339–3347.4.Bowman,C.C.andClements,J.D.,et al.(2001).Infec. Immun. 69,1528–1535.5.Tinker,J.K.,et al.(2003).Infec. Immun. 71,4093–4101.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20372 Immobilized D-GalactoseSupport:6%beadedagaroseCapacity:≥20mgjacalin/mLresin
5mL $145
Immobilized Boronic Acid
For ribonucleoside isolation.
B
OH
OHHN
Thermo Scientific Immobilized Boronic Acid
OOH
Resi
nB
ead
ReferencesVlassara,H.,et al.(1981).Proc. Natl. Acad. Sci. USA78,5190–5192.Gehrke,C.W.,et al.(1978).J. Chromatogr.150,455–476.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
20244 Immobilized Boronic AcidSupport:PolyacrylamideresinbeadsCapacity:≥99%bindingandrecoveryof110μmol AMP/mLofresinSpacer:m-aminophenylgroupLoading:100μmolboronate/mLofresin
10mL $190
80 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce
Thermo Scientific MagnaBind Beads and Supports
A convenient method for isolating biomolecules using affinity binding, while retaining biological activity.
Magneticbeadsareaconvenientaffinitysupportforavarietyofassays,whichalloweasypurificationofthetargetwithoutcolumnsorcentrifugation.Afterabindingstepinanaffinitypurificationprocedure,themagneticparticlesareeasilyandrapidlycollectedbyplacingthemicrocentrifugetubeorreactionvesselnextanappropriaterare-earthmagnet(Figure1).ThermoScientificMagnaBindbeadsrespondrapidlytoMagnaBindMagnetsbutcanbeeasilydispersedandregatheredmultipletimes(i.e.,theywillnotirreversiblyaggregate)becausetheydonothaveanymagneticmemory.MagnaBindBeadsareavailablepre-coatedwithProteinA,ProteinG,streptavidin,anti-mouseoranti-rabbitantibodies.Activatedbeads,witheitheramineorcarboxylgroups,arealsoavailableforattachingotherproteinsoraffinityligandstoamagneticparticle.
Highlights:•Availablepre-coatedwithpopularaffinityligandsorderivatized forcovalentattachmentofproteinsandotherspecificligands•Beadsdonotirreversiblyaggregatebecausetheyhaveno magneticmemory;collectanddispersethebeadsmultipletimes ifneeded•Mostseparationsrequireashortfive-to10-minutebench-top procedure
Applications:•Cellsortingusingpositiveornegativeselection•Proteinpurificationorimmunoassaysusingdirectorindirectmethods
Characteristics of underivatized Thermo Scientific MagnaBind Beads.
Composition Silanizedironoxide
Magnetization 25–35EMU/g
Type of Magnetization Superparamagnetic(nomagneticmemory)
Surface Area >100m2/g
Settling Rate 4%in30minutes
Effective Density 2.5g/mL
Number of Beads 1x108beads/mg
pH Stability Aqueoussolution,abovepH4.0
Concentration 5mg/mL
Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with antibiotic medium or g-irradiated.
Figure 1. Thermo Scientific MagnaBind Magnet for 1.5mL Microcentrifuge Tube. ThermoScientificMagnaBindBeadsinsolutionswithinamicrocentrifugetubearerapidly“pelleted”whenthetubeisplacedinthemagnetizedholder.Magnetsforsixmicrocentrifugetubesand96-wellmicroplatesarealsoavailable.
ReferencesChaudhuri,T.K.,et al.(2001).Cell107,235–246.Newey,S.E.,et al.(2001).J. Biol. Chem.276,6645–6655.Xu,X.,et al.(2001).J. Biol. Chem.276,43221–43230.
Ordering Information Product #
Description
Pkg. Size
U.S. Price
21344 MagnaBind Streptavidin Beads 5mL $271
21348 MagnaBind Protein A Beads 5mL $325
21349 MagnaBind Protein G Beads 5mL $325
21354 MagnaBind Goat Anti-Mouse IgG Beads 50mL $256
21356 MagnaBind Goat Anti-Rabbit IgG Beads 50mL $256
21353 MagnaBind Carboxyl Derivatized Beads 5mL $127
21352 MagnaBind Amine Derivatized Beads 5mL $130
21358 MagnaBind Magnet for 96-Well Plate Separator
1magnet $392
21357 MagnaBind Magnet for 1.5mL Microcentrifuge Tube
1magnet $152
21359 MagnaBind Magnet for 6 x 1.5mL Microcentrifuge Tubes
1magnet $305
Thermo Scientific Affinity Supports
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 81
ÄKTA, ÄKTApurifier™, HiTrap® and pGEX® are trademarks of GE Healthcare.Phos-Select™ is a tradmark of Sigma-Aldrich Co.NanoLC™ is a trademark of Eskigent.Trisacryl® is a trademark of Pall Corporation.Luer-Lok™ is a trademark of Becton, Dickinson and Company.Brij® is a registered trademark of ICI Americas.Cibacron® is a registered trademark of Ciba Specialty Chemicals, Inc.Triton® is a registered trademark of Rohm & Haas Company.Tween® is a trademark of ICI Americas.
Contact Information
Belgium and Europe, the Middle East and Africa Distributors Tel: +32 53 85 71 84
France Tel: 0 800 50 82 15
The Netherlands Tel: 076 50 31 880
Germany Tel: 0228 9125650
United Kingdom Tel: 0800 252 185
Switzerland Tel: 0800 56 31 40
Email: [email protected] www.thermoscientific.com/perbio
United States Tel: 815-968-0747 or 800-874-3723 Customer Assistance E-mail: [email protected] www.thermoscientific.com
© 2010 Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Unless indicated otherwise on the inside back cover, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. 16
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