TALON Metal Affinity Resins User Manual - … · 2017-09-12 · Using theTALON® Metal Affinity...
Transcript of TALON Metal Affinity Resins User Manual - … · 2017-09-12 · Using theTALON® Metal Affinity...
TALON®
Metal Affinity ResinsUser Manual
PT1320-1 (PR6Z2142)Published 25 April 2007
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 2 Version No. PR6Z2142
TALON® Metal Affinity Resins User Manual
Table of Contents
I. Introduction 4
II. List of Components 13
III. Additional Materials Required 15
IV. Buffers for TALON® Purification & Buffer Kits 20
V. Buffers for TALON® Magnetic Beads 21
VI. Transformation & Protein Expression 22 A. TransformationofHostCellswithExpressionVectors 22 B. ProteinExpression 22
VII. Sample Preparation 23
A. TALON®xTractorBufferSamplePreparation 23
B. StandardSamplePreparationtoIsolateNativeProteins 23
C. StandardSamplePreparationtoIsolateDenaturedProteins 24
D. StandardSamplePreparationforTALON®CellThruResin 25
E. StandardHT96-WellSamplePreparation 26
F. StandardSamplePreparationforTALON®MagneticBeads 26
G. SamplePreparationDirectlyfromOvernightCulturesfor
TALON®MagneticBeads 27
VIII. Protein Purification Protocols 28
A. GeneralInformation 28
B. Batch/Gravity-FlowColumnPurification 30 TALON®Resin,SuperflowResin,orCellThruResin
C. Large-ScaleBatchPurification 32 TALON®Resin,SuperflowResin,orCellThruResin
D. Medium-PressureColumnPurification 33 TALON®SuperflowResin
E. 5mlTALON®SingleStepColumnPurification 34
F. 20mlTALON®SingleStepColumnPurification 36
G. TALONspin™ColumnPurification 38
H. TALON®HT96-WellPurificationProtocol 40
I. TALON®MagneticBeadsPurificationProtocol 43
IX. Resin Washing, Reuse, and Storage 46
X. Troubleshooting Guide 48
XI. References 53
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Table of Contents continued
Notice to Purchaser
Clontechproductsaretobeusedforresearchpurposesonly.Theymaynottobeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,in vitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.
LicensedunderU.S.PatentNo.4,569,794anditsinternationalequivalentsforuseinresearchrelatedbiopolymers.Licenses forcommercialapplicationsareavailable fromIndianaPro-teomicsConsortium,Inc.(Inproteo).
TALON®ResinproductsarecoveredunderU.S.PatentNo.5,962,641.
Sepharose®isaregisteredtrademarkofGEHealthcare.
Triton™isatrademarkofTheDowChemicalCompany.
Superflow™,Uniflow™,andCellThru™aretrademarksofSterogeneBioseparations,Inc.Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.,unlessnotedotherwise.ClontechisaTakaraBioCompany.©2007
Appendix A. ReagentCompatibilitiesandIncompatibilities 54
Appendix B.Mini-ScaleProteinPurificationProtocolforTALON®or TALON®SuperflowResin 56
Appendix C.VectorInformation 58List of Figures
Figure1. SchematicdiagramoftheTALON®IMACSystem. 5
Figure2. Elutionmechanismofrecombinantpolyhistidine-taggedproteinsfromTALON®Resin. 6
Figure3. BindingofhistidinestotheTALON®Resinmetalion. 6
Figure4. UsingtheTALON®MetalAffinityResinsUserManual 7
Figure5. Purificationofpolyhistidine-taggedproteinsusingTALON®Resin 19
Figure6. pHAT10/11/12combinedvectormapandMCS. 58
Figure7. pHAT20combinedvectormapandMCS. 59
List of TablesTableI. ProteinpurificationusingTALON®Resins. 10
TableII. TALON®Resincharacteristics 12
TableIII. Reagentcompatibility 54
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I. Introduction
Proteinshaveevolvedverycomplexstructuresinordertoperformadiversear-rayoffunctions.Asaresult,theirphysicochemicalpropertiesvarygreatly,pos-ingdifficultiesfordevelopingversatilepurificationprotocols.Onewaytocir-cumventthisproblemistoincorporateapurificationtagintotheprimaryaminoacidsequenceofatargetprotein,thusconstructingarecombinantproteinwithabindingsitethatallowspurificationunderwell-defined,genericconditions.
Immobilized Metal Affinity Chromatography (IMAC)IMACwasintroducedin1975asagroup-specificaffinitytechniqueforsepa-ratingproteins(Porathet al.,1975).Theprincipleisbasedonthereversibleinteractionbetweenvariousaminoacidsidechainsandimmobilizedmetalions.Dependingontheimmobilizedmetal ion,differentsidechainscanbe involved in theadsorptionprocess.Mostnotably,histidine,cysteine,andtryptophansidechainshavebeenimplicatedinproteinbindingtoim-mobilizedtransitionmetalionsandzinc(Figure1,Porath,1985;Sulkowski,1985;Hemdan&Porath,1985a;Hemdan&Porath,1985b;Zhaoet al.,1991).
TALON® IMAC ResinsTALON®Resinsaredurable,cobalt-basedIMACresinsdesignedtopurifyrecombinantpolyhistidine-taggedproteins(Bushet al.,1991).Theseresinsarecompatiblewithmanycommonlyusedreagents(AppendixA),andallowpro-teinpurificationundernativeordenaturingconditions.Theycanbeusedwithallprokaryoticandeukaryoticexpressionsystemsinavarietyofformats,in-cludingsmall-(ormini-)scalebatchscreening,large-scalebatchpreparations,andmethodsusinggravity-flowcolumnsandspincolumns.Inaddition,pro-tocolsusedwithNi+2-basedIMACcolumnsusuallyworkwithTALON®resins.
TALONMagneticBeadsareagarosebeadsutilizingourpatentedTALONtech-nology.ThebeadscombinetheadvantageofhighlyselectiveTALONchemistrywithmagneticbeadseparation.Magneticparticlesinthebeadsfacilitatequickandeasypurificationofproteinsatmicroscalelevelusingamagneticseparator.MicroscalepurificationwithTALONMagneticBeadscanbeusedforscreeningofexpressionlevelsorforprotein-proteininteractionstudies.
Tetradentate metal chelatorToovercometheproblemofmetalleakageencounteredwithotherIMACres-ins,TALON®Resinutilizesaspecialtetradentatemetalchelatorforpurifyingrecombinantpolyhistidine-taggedproteins(U.S.PatentNo.5,962,641).Thischelatortightlyholdstheelectropositivemetalinanelectronegativepocket(Figure1),whichisidealforbindingmetalionssuchascobalt.ThebindingpocketisanoctahedralstructureinwhichfourofthesixmetalcoordinationsitesareoccupiedbytheTALONResinligand.ThisprocessenhancestheproteinbindingcapacityofTALONResinbymakingtheboundmetalionac-cessibletosurroundingpolyhistidine-taggedproteins.Thetetradentatemetalbindingmeansthatnometallossoccursduringproteinpurificationunderrec-
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ommendedconditions,eveninthepresenceofstrongdenaturantssuchas6Mguanidinium.SuchdurabilityallowsTALONResintobereused(SeeSectionIX).
Cobalt IMAC Resin permits milder elution conditionsTALONResinexhibitssubtleyetimportantdifferencesinbindingofpolyhis-tidine-taggedproteinswhencomparedwithnickelIMACresins.Forexample,nickel-basedIMACresinsoftenexhibitanundesirabletendencytobindun-wantedhostproteinscontainingexposedhistidineresidues(Kasheret al.,1993).WhileTALONResinbindspolyhistidine-taggedproteinswithenhancedselectivityovernickel-basedresins,itexhibitsasignificantlyreducedaffin-ityforhostproteins.Thisbehaviorofferstwopracticaladvantages.First,virtuallynobackgroundproteinsareboundtotheresinwhenthesampleisapplied;consequently,cumbersomewashingproceduresarenotgenerallyrequiredbeforeproteinelution.Second,polyhistidine-taggedproteinselutefromtheresinunderslightlylessstringentconditions—aslightlyhigherpHor lower imidazoleconcentration—thanwithnickel IMACresins.Elutionoccurswhentheimidazolenitrogen(pKaof5.97)isprotonated(Figure2),generatingapositivelychargedammoniumion,whichisrepelledbythepositivelychargedmetalatom.Alternatively,theboundpolyhistidine-taggedproteincanbecompetitivelyelutedbysimplyaddingimidazoletotheelu-tionbuffer,becauseimidazoleisidenticaltothehistidinesidechain.
Figure 1. Schematic diagram of the TALON® IMAC System.Part A.TALONMetalAffinityResin;ASepharosebeadbearingthetetradentatechelatoroftheCo2+metalion.Part B.Thepolyhi-stidine-taggedrecombinantproteinbindstotheresin.
I. Introduction continued
HC
N
N
N
N
N
H2C
H2C
COO–
COO–
COO–
Co2+SepharoseBead
A B
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Polyhistidine affinity tagsHistidinesexhibithighlyselectivecoordinationwithcertaintransitionmet-alsandhavegreatutilityinIMAC.UnderconditionsofphysiologicalpH,histidinebindsbysharingelectrondensityoftheimidazolenitrogenwiththe electron-deficient orbitals of transition metals (Figure 3). Althoughthree histidines may bind transition metals under certain conditions,six histidines reliably bind transition metals in the presence of strongdenaturants such as guanidinium (Hochuli et al., 1987). Such proteintags are commonly referred to as“6 x histidine,”“hexaHis,” or“(His)6.”
HAT—a novel IMAC affinity tag
With the advent of recombinant genetic technologies, the design andproduction of recombinant proteins containing novel polyhistidine tagsontheirN-orC-terminihasbecomemorestraightforward(Hochuliet al.,1987;Hochuliet al.,1988).TheHATsequence(patentpending)isanovelIMACaffinitytagderivedfromauniquenaturalproteinsequence(Chagaet al.,1999).Itcontainssixhistidinesunevenlyinterleavedbyotheraminoacidresidues(seeAppendixC).TheHATaminoacidsequenceisderivedfromtheN-terminusofchickenmusclelactatedehydrogenase—asequencethatisuniqueamongreportedproteinsequences.Thenoveltagdoesnothave theexcessivepositivechargecharacteristicof thecommonlyused6xhistidinetag,thuscontributingtobettersolubilityofHAT-fusionpro-teinsandsimilaraffinity towards immobilized transitionmetal ionsandzinc.ClontechofferstheHATProteinExpressionandPurificationSystem(Cat. No. 631205)—a complete system containing reagents and vectors
Figure 2. Elution mechanism of recombinant polyhistidine-tagged proteins from TALON® Resin. Elutionoccurswhentheimidazoleni-trogen(pKa=5.97)isprotonated,generatingapositivelychargedammoniumionwhichisrepelledbythepositivelychargedmetalion.Alternatively,theboundpolyhistidine-taggedproteincanbecompetitivelyelutedbyaddingimidazoletotheelutionbuffer.
Figure 3. Binding of histidines to the TALON® Resin metal ion.UnderconditionsofphysiologicalpH,histidinebindsbysharingimidazolenitrogenelectrondensitywiththeelectron-deficientorbitalsofthemetalion.
I. Introduction continued
N
N
N
N
2 +
H +
N
N H
? Unprotonated Histidine
binds to metal
N
N H
H
Protonated Histidinerepelled by metal
+
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Protein Purification Strategy (Section I) • Protein Purification Methods - Resin Characteristics • Choosing Buffers • Elution Strategy
Buffers (Sections III & IV) Native Denaturing
Protein Expression (Section VI) A. Transformation B. Protein Expression
Sample Preparation (Section VII)
Native Purification Denaturing PurificationA. xTractor Buffer C. Standard &B. Standard & Superflow Resin Superflow Resin D. CellThru ResinD. CellThru Resin F. TALON Magnetic E. High-throughput (96-well) BeadsF. & G. TALON Magnetic Beads
Protein Purification (Section VIII) B. Batch or Gravity FlowC. Large-Scale Batch
D. Medium-Pressure & FPLC Column E. 5 ml Single Step ColumnsF. 20 ml Single Step Columns
G. TALONspin™ Columns H. HT 96-Well Plate I. TALON Magnetic Beads Appendix B. Mini-Scale
Figure 4. Using the TALON® Metal Affinity Resins User Manual.Overviewoftheprocedures.
I. Introduction continued
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designedforbacterialexpressionandpurificationofHAT(histidineaffinitytag)proteins.Eachofthethreevectors—pHAT10,pHAT11,andpHAT12—containamultiplecloningsite(MCS)inallthreeframestoallowcloningoftargetcDNA.(ForvectormapandMCS,seeAppendixCofthisUserManual.)AconvenientlylocatedenterokinaseproteolyticsitebetweentheHATsequenceandtheMCSprovidesameansforremovingtheaffinitytag.
Formoreinformation,seetheHATExpression&PurificationSystemUserManual(PT3250-1),whichcanbedownloadedfromourwebsiteatwww.clontech.com.
TALON® Express Bacterial Expression and Purification Kits
TALONExpressBacterialExpressionandPurificationKits aredesignedforthecloning,expression,andpurificationofpolyhistidine-taggedproteinsusing E. coli.ThekitscontaintwoseparatebacterialexpressionvectorsencodingN-orC-terminal6xHNfusiontags.TheseIPTG-inducible,pET-basedvectorsprovidehighlevelsofproteinexpression.TheexpressedproteinsarereadyforquickandeasypurificationusingtheTALONresinandbuffersprovidedinthekits.
Overview of TALON® Resins
Thefollowingisalistofdifferentresinformatstomeetyourpurificationneeds.
• TALON® Metal Affinity Resin is useful for batch and low-pressurechromatographicapplications.This resinutilizesSepharoseCL-6B (GEHealthcare),adurablesubstrate thatperformsverywellundernativeanddenaturingconditionsincentrifuge-mediatedpurificationschemes.Thelargeporesizeresinhasahigh-bindingcapacity.Thisresinisalsoavailablepre-packedin2mlgravitycolumns.
• TALON® Superflow Resinisusefulforarangeofapplications,includingmediumpressureapplicationswithFPLCsystemsatbackpressuresofupto150psi(1MPa)andhighflowratesupto5mlpercm2permin.Thisresinisrecommendedifshortpurificationtimesareessential,orifpurificationprotocolsdevelopedatbenchscalewillbescaledupforlargervolumes.
ThisresinutilizesSuperflow-6(SterogeneBioseparations,Inc.),anaga-rose-basedmediumfeaturingauniquepolysaccharidecompositionthatresistsbiologicaldegradation.Superflow-6beadsarealsostabilizedbyachemicalcross-linkingreactionthatallowsflowratesupto10timeshigherthanarepossiblewithregularcross-linkedbeads.
TheTalon®SuperflowResinisalsopresentinthehighthroughput(HT)96-wellplate.
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• TALON® CellThruResinisanovelIMACresinforpurifyingpolyhistidine-taggedproteinsfromcrudecelllysates,sonicates,andfermentationliquids.ThelargerbeadsizeofTALONCellThruResin(300–500µm)permitscellulardebristoflowthroughthecolumn,eliminatingtheneedforhigh-speedcentrifugation.DestabilizingfactorsareremovedmorequicklywiththisresinthanwithotherIMACresins,becausethenumberofstepsarereduced.
CellThru2ml&10mlDisposableColumnshavealargefilterporesize(90–130µm)thatallowscellulardebristoflowthroughthecolumnduringthepurificationprocess.The2mlcolumnsaresuitablefor1–2mlbedvolumes,whilethe10mlcolumnsaresuitablefor5–10mlbedvolumes.
• TALONspin™ Columns areidealforrapidlyandsimultaneouslypurifyingsmallamountsofpolyhistidine-taggedproteins.Thesecolumnsarerecom-mendedforsingle-useapplicationsorforuseasminigravity-flowcolumns.Eachcolumncontains0.5mlofTALON-NXResin,whichisoptimizedforperformanceinaspincolumn.Eachcolumnwillyield2–4mgofpolyhi-stidine-taggedprotein;exactyieldswillvarywithconditionsusedandpolyhistidine-taggedproteincharacteristics.Inaddition,yieldandpuritywilldependuponexpressionlevelandlysateconcentration.Beginningwiththeclarifiedsample,theentireproceduretakesapproximately30min.
• TALON® Magnetic Beads areusefulformicroscalepurificationofpolyhisti-dine-taggedproteinsundernativeordenaturingconditions.Thebeadscanalsobeusedtopurifyproteinsdirectlyfromcleared(centrifuged)orcrudecelllysates.Forscreeningofexpressionlevels,proteinscanbepurifieddirectlyfromovernightculturesassmallas0.5ml(dependingontheexpres-sionlevel).TheuseofTALONchemistryallowsforseamlessscaling-uptolarge-scalepurificationoftargetproteinsusingourstandardTALONresin.
TALONMagneticBeadsaresuppliedasa5%suspensionin25%ethanol,availableineithera2x1mlor6x1mlformat.Thebeadshaveabindingcapacityof750µgof6xHN-taggedAcGFPper1mlofsuspension.Whenperformingassaysinsingletubes,100–200µlofbeadsaresufficientforeachassay.Smalleramountsofbeadsmaybeused,buttheremaybedifficultiesinhandlingthebeadsinsmallbuffervolumes.
I. Introduction continued
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TABLE I. PROTEIN PURIFICATION USING TALON® RESINS
Method Application Key Benefit
TALON®Metal Affinity Resin or TALON® Superflow ResinMini-Scale •Checkforpresenceoftaggedprotein•Fast(AppendixB) •Estimateexpressionlevels •Requiresonly1mlofcell •Testbufferconditions culture+1mlofresin
Batch/Gravity •Purify>5mgoftaggedprotein •VeryhighpurityFlow Column using1mlofresin •Doesnotrequire(Sec.VIII.B) pressurizedcolumn equipment
Large-Scale •Large-andproduction-scale •Fasterthanprotocolsthat(Sec.VIII.C&D) purification;easytoscaleup usegravity-flowcolumns •Higherpuritythanusing batchprocessaloneTALON® CellThru ResinBatch/Gravity •Forpurifyingproteinsfrom •FastFlow Column & nonclarifiedcelllysates,sonicates, •Doesnotrequirehigh-Large-Scale orfermentationliquids speedcentrifugation(Sec.VIII.B&C) TALON® Single Step Columns (5 ml, 20 ml) Miniprep •Processseveraldifferentsamples •Fast(~30–40min)1
(Sec.VIII.E&F) simultaneously •Usesunlysedcellculture •Lysebacterialcellsandbind •Simplifiesscreeningof histidine-taggedproteininonestep multipleproteins •Obtain0.2–0.6mg(5mlcolumn) •Ready-to-usecolumns or0.5–4mg(20mlcolumn).TALONspin™ Columns Spin Column •Processseveraldifferentsamples •Fast(~30min)2
(Sec.VIII.G) simultaneously •Usesonly0.6–1mlof •Obtain2–4mgofpurifiedprotein cellculturelysate perspincolumn •Ready-to-usecolumnsTALON® HT 96-Well Plates96-Well Plates •High-throughputprocessingofsamples•Fast(<30min)2
(Sec.VIII.H) •Obtainupto1.0mgofpurifiedprotein•Usesupto2mlof perwell crudelysateperloadTALON®Magnetic BeadsMagnetic Beads•Microscalepurification •Fast(Sec.VII.F&G •Checkforpresenceoftaggedprotein •Requires0.5mlcultureandSec.VIII.I) •Estimateexpressionlevels •Doesnotrequirehigh- •Purifyfromcrudeuncleared speedcentrifugation celllysatesorcultures •Amenabletohigh-throughput1Includestimeforsampleprepandpurification.2Startingwithclarifiedlysate;doesnotincludetimetopreparesamples.
I. Introduction continued
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Protein Purification Methods Using TALON® ResinThefollowinggeneralguidelinesareusedforpurifyingpolyhistidine-taggedproteinfromtransformedE. colicultures.Figure4andTableIprovideanoverviewofTALONResinproteinpurificationmethodsandapplications.Chooseamethodthatbestsuitsyourresearchneeds.
• Use2mlof resinsuspensionper~3mgofanticipatedpolyhistidine-taggedprotein.2mlofhomogeneouslyresuspendedresinwillprovide1ml(bedvolume)ofTALONResin.
• Thebuffersandpurificationconditionsshouldworkwellformostsoluble,monomericproteinsexpressedinE. coli.
• Initially,testeachdifferentexpressionsystemandpolyhistidine-taggedproteininsmall-scalebatchpurificationtodetermineexpressionlevelsandtooptimizetheprotocol.TALONSingleStepColumnsaredesignedforthistypeofanalysis(SectionVIII.E&F).Alternatively,Amini-scalebatchpurificationprotocolisprovidedinAppendixB;oryoucanuseaTALONspinColumn(SectionVIII.G).
• Purificationmethodsthatworkwithnickelorzinc-basedIMACresinsshouldalsoworkwiththeseresins.However,someoptimizationmayberequired.
Note:TALONresinhasbeenoptimizedandshouldonlybeusedwiththebufferformula-tionsoutlinedinthisusermanualforoptimalperformance.
Choosing the Buffers: Imidazole Versus pH Gradient
TALONResinpurificationschemestypicallyuseeitheranimidazoleorapHgradientforwashingandelution.ImidazoleintheEquilibrationand/orEquilibration/WashBuffersminimizesnonspecificbindingandreducestheamountofcontaminatingproteins.Thus,werecommendfirstpurifyingpoly-histidine-taggedproteinsusinganimidazolegradient.However,imidazoleandpolyhistidine-taggedproteinsabsorbat280nmandelutionpeaksmaybedifficulttodetectspectrophotometrically,especiallyifyouarepurifyingsmallamountsofpolyhistidine-taggedproteins.Inthesecases,collecttheleadingedgeoftheimidazolebreakthroughpeakandcheckforpolyhistidine-taggedproteinsbyaproteinspecificassay(Bradford,1976)andSDS/PAGE.Alternatively,useapHgradienttopurifypolyhistidine-taggedproteinsthatarestablefrompHrange5.0–7.0.SeeSectionIIIforbuffercompositions.
Elution Strategy: Step Versus Linear Gradients Inmostcases,stepgradientsarepreferredoverlineargradients,becauselineargradientsleadtobroadelutionpeaks,whichcandilutetheproductandmakedetectiondifficult.Scaling-upstepgradientsisalsolesscompli-catedthanscaling-uplineargradients.
I. Introduction continued
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TABLEII.TALON®RESINCHARACTERISTICS
TALON® TALON® TALON® TALONspin™Feature Resin Superflow CellThru Column
Capacity1 5–15 5–20 5–10 2–4(mgprotein/mlresin)
Matrix SepharoseCL-6B Superflow Uniflow Sepharose6B
Beadsize(µm) 45–165 60–160 300–500 16–24
Max.Linear 75–150 3,000 800 n/a2flowrate(cm/hr)
Max.Volume 0.5–1.0 50 13 n/aflowrate3(ml/min)
Max.Pressure 2.8psi 140psi 9psi n/a 0.2bar 10bar 0.62bar n/a 0.02MPa 0.97MPa 0.06MPa n/a
pHstability 2–14(2hr) 2–14(2hr) 2–14(2hr) 2–8.5(2hr) 3–14(24hr) 3–14(24hr) 3–14(24hr) 2–7.5(24hr)
Proteinexclusion 4x107 4x106 2x107 n/alimit(Da)1Thebindingcapacityforindividualproteinsmayvary.EachoftheabovementionedTALONproductshas
differentapplications.PleaserefertoTableIforapplicationsandbenefits.2n/a=notapplicable3Determinedona5x1cmcolumn.
I. Introduction continued
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II. List of Components
TALONResin,TALONSuperflowResin,andTALONCellThruResinaresuppliedas50%(w/v)slurriesinnonbuffered20%ethanol.Pleasenotethatduringshippingandstorage,theresinwillsettle;thus,werecommendthatyouthoroughlyresuspenditbeforealiquotting.2mlofhomogeneouslyresuspendedresinwillprovide1mlofTALONResinwithabindingcapacityofatleast5mgofpolyhistidine-taggedprotein.
Store all of these resins, columns and buffers at 4°C unless otherwiseindicated.Do not freeze TALON® Resins.
• TALON® Metal Affinity Resin Cat. No. Amount 635501 10ml 635502 25ml 635503 100ml 635504 250ml
• TALON® Superflow Resin Cat. No. Amount 635506 25ml 635507 100ml
• TALON®Single Step Columns (5ml,Cat.Nos.635628&635631; &20ml,Cat.No.635632) ThesecolumnscontainadrymixtureofTALONCellThruresin andxTractor
Buffertoextractandbindhistidine-taggedproteinsinonestep.
• TALONspin™Columns (Cat.Nos.635601,635602,635603) Thesecolumnscontain0.5mlofTALON-NXresin asa50%suspension
innonbuffered20%ethanol.
• TALON® HT 96-Well Plate (Cat.No.635622) 1 TALON96-WellPlate 1 PlateTopSeal 1 PlateBaseSeal 1 CollectionPlate
• TALON® Magnetic Beads(Cat.Nos.635636&635637) Cat. No. Amount 635636 2x1ml 635637 6x1ml
• TALON® Magnetic Beads Buffer Kit (Cat.No.635638) 60ml 5XEquilibration/WashBuffer 15ml 4XElutionBuffer 30ml 1XxTractorBuffer
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II. List of Components continued
• TALON® CellThru Resin Cat. No. Amount 635509 10ml 635510 100ml
• TALON® CellThru Disposable Columns Cat. No. Size 635512 50x2mlcolumn 635513 20x10mlcolumn
• TALON® 2 ml Disposable Gravity Columns (Cat.No.635606)
• TALON® Purification Kit(Cat.No.635515)
10ml TALON®MetalAffinityResin 160ml 5XEquilibration/WashBuffer (250mMsodiumphosphate,1.5MNaCl,pH7) 160ml 5XEquilibrationBuffer (250mMsodiumphosphate,1.5MNaCl,pH8) 25ml 10XElutionBuffer (1.5Mimidazole,pH7) 5 2mlDisposableGravityColumns 1 10mlDisposableGravityColumn
• TALON® Buffer Kit (Cat.No.635514)
160ml 5XEquilibration/WashBuffer (250mMsodiumphosphate,1.5MNaCl,pH7) 160ml 5XEquilibrationBuffer (250mMsodiumphosphate,1.5MNaCl,pH8) 25ml 10XElutionBuffer (1.5Mimidazole,pH7)
• TALON® xTractor Buffer Kit (Cat.No.635623)
StoreDNaseIat–20°C.Ifaprecipitatehasformedinthelysozymesolution,allowthetubetowarmatroomtemperatureandgentlyinvertthetube.Thesolutionmayremainturbidafterthisprocedure.
200ml 1XxTractorBuffer 2.5ml 50XLysozyme 400µl DNase(1unit/µl)
• TALON® xTractor Buffer (Cat.No.635625) 500ml 1XxTractorBuffer
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See Section IV for preparing buffers with theTALON® Purification Kit(Cat.No.635515)ortheTALON®BufferKit(Cat.No.635514).Ifyouhave not purchased thosekits,werecommendpreparingthefollowingbuffersforpurifyingpolyhistidine-taggedproteinsundernativeordenaturingcondi-tions.Beforepreparingotherbuffercompositions,pleaseconsultAppendixAtoevaluateresincompatibility.ForTALONMagneticBeads(seeSectionV),usethesameEquilibration/Wash Bufferaswiththeresins,andelutewithanimidazole-basedelutionbuffercontainingahigherconcentrationofimidazolethanthatusedtoelutefromtheresins,asdescribedinSectionV.D.
Choosing Buffers
Todecreasetheamountofnonspecificallyboundproteins,werecommendusingtheEquilibration/Wash BufferatpH7.0duringpurification;however,ifyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbatpH7.0,usetheEquilibrationBufferatpH8.0(inplaceoftheEquilibration/WashBuffer)duringallextractionandwashsteps.NotethatatelevatedpHvalues,aminoacidsotherthanhistidine,aswellasthepeptidebond,contributetoproteinadsorption.Thus,proteinswithoutapolyhistidinetagcanalsoadsorbtoIMACresins,whichdecreasesresincapacityandthefinalpurityofyourtargetprotein.Youmaychoosetouseeithernativeordenaturingbuffer conditions, depending on the solubility of your protein. Figure 5outlinesthepurificationprocedure.
A. Native Buffers
Nativeproteinpurificationregimensusebufferconditionsthatpreservethenative,three-dimensionalstructureandsurfacechargecharacteris-ticsofaselectedsolubleproteinduringharvestfromanexpressionhost.ThelowaffinityofTALONResinfornonpolyhistidine-taggedproteinsminimizescontaminantcarryover.Inaddition,increasingbufferionicstrengthcanminimizenonspecificinteractions.Regardlessofthecon-ditionsusedandthenatureofthepolyhistidine-taggedproteinbeingpurified,mostapplicationswillbenefitfromthepresenceof100–500mMNaClintheIMACbuffer.Inmanycases,addingglycerolorethyleneglycolneutralizesnonspecifichydrophobicinteractions.Smallamountsof nonionic detergent may also dissociate weakly bound species.
• 1X Equilibration/Wash buffer (pH7.0)* 50mMsodiumphosphate 300 mM NaCl • 1X Equilibration buffer (pH8.0)* 50 mM sodiumphosphate 300 mM NaCl
III. Additional Materials Required
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III. Additional Materials Required continued
• 1X Elution buffer* – Imidazole Elution (pH7.0) – pH Elution (pH5.0)1
50mMsodiumphosphate 50mMsodiumacetate 300mMNaCl 300mMNaCl 150mMimidazole 1Preparefreshbeforeuse.
• HT 96-Well Plate Wash buffer (pH7.0)* 83mMsodiumphosphate 500mMNaCl 10mMimidazole
• TALON Magnetic Beads 1X Elution buffer* – Imidazole Elution (pH7.0)
50mMsodiumphosphate 300mMNaCl 250mMimidazole
B. Denaturing Buffers Denaturants, such as 6 M guanidinium, enhance protein solubility.
Because proteins overexpressed in prokaryotic systems are some-times insoluble,youmayneedtopurifyproteinsunderdenaturingconditions.Whenpurifyingproteinsunderdenaturingconditions,werecommendpreparingthebuffersindicatedbelow.
Inthepresenceof6Mguanidinium,the resin’s color will change from a pinkish-mauve to violet due to a change in metal ion hydration in response to the chaotrope.Afterremovalofthechaotrope,theresinwillreturntoapinkish-mauvecolor.Thechangetovioletdoesnotreflectanychangeinthephysicalorchemicalpropertiesoftheresin.Infact,thecolorchangecanbeusefulforindicatingthebufferinwhichtheresinissuspended,andforfollowingthemovementofguanidiniumthroughtheresinbed.
• 1X Equilibration/Wash Buffer (pH7.0)* 50 mM sodiumphosphate 6 M guanidine-HCl 300 mM NaCl
• 1X Equilibration Buffer (pH8.0)* 50 mM sodiumphosphate 6 M guanidine-HCl 300 mM NaCl
*SeeSambrook,AppendixB.21,oryourstandardprotocolforpreparingsodiumphosphatebuffer.
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III. Additional Materials Required continued
• 1X Imidazole Elution Buffer (pH7.0) 45 mM sodiumphosphate 5.4 M guanidine-HCl 270 mM NaCl 150 mM imidazole
• TALON Magnetic Beads 1X Elution Buffer* – Imidazole Elution (pH7.0)
45mMsodiumphosphate 5.4 M guanidine-HCl 270mMNaCl 250mMimidazole
C. Additional Buffers & Reagents
• MES Buffer 20mM2-(N-morpholine)-ethanesulfonicacid(MES),pH5.0 • 5X SDS PAGE sample buffer 15% β-Mercaptoethanol(β-ME) 15% SDS 50% Glycerol 1.5% Bromophenolblue
• Imidazole (Sigma,Cat.No.I0250)AlsosuitableforFPLCapplications • Bio-Rad Protein Assay (Bio-Rad,Cat.No.500-0001)
D. Additional Materials required for TALON® CellThru Resin
• CellThru2mlDisposableColumns(Cat.No.635512) • CellThru10mlDisposableColumns(Cat.No.635513)
E. Additional Materials for TALON® HT 96 Plate Vacuum Purification •QIAVac96(QIAGEN,Cat.No.19504),NucleoVac(MACHEREY-NA-
GEL,Cat.No.740630),orsimilarvacuummanifold •(Extra)Collection96-DeepWellTiterPlates(WhatmanCat.No.7701-
5200orEvergreenCat.No.240-8556-030)
Centrifugation • Centrifugewitharotorforcentrifugationofmicrotiterplates,such
astheAllegra6RCentrifuge(BeckmanCoulter)withtheGH3.8;GH3.8A;orJS4Beckmanrotors.
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III. Additional Materials Required continued
F. Additional Materials for TALON® Single Step Columns •1XEquilibration/WashBuffer*(50mMsodiumphosphate,
300mMNaCl,pH7.0) •Wash-2Buffer*(50mMsodiumphosphate,300mMNaCl,
7.5mMimidazole,pH7.0) •1XElutionBuffer*(50mMsodiumphosphate,300mMNaCl,
150mMimidazole,pH7.0) •15mlscrew-captubes(5mlcolumns)or50mlscrew-captubes(20
mlcolumns),receivingtubesforfractionstorage •[Optional]BCAProteinAssayKit(Pierce,CatNo.23226)
*ThebuffersusedfortheSingleStepColumnscanbepreparedbydilutionofthebuf-fersinourTALONBufferKit(Cat.No.635514).SeeSectionIVforpreparationdetails.
G. Additional Materials for TALON® Magnetic Beads •Magneticseparator(colorlessorwhiteforbestvisibility,sincethe
beadsareblack) •1.5mland0.5mlmicrofugetubes •DNaseI
Note:AlthoughTALONxTractorBufferisincludedintheTALONMagneticBeadsBufferKit(Cat.No.635638),ifmoreisneeded,itisavailableasCat.No.635623.
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III. Additional Materials Required continued
Figure 5. Purification of polyhistidine-taggedproteins using TALON®Resin. TheprotocolsinthisUserManualaredesignedusingtheEquilibration/WashBufferatpH7.0.IfyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbatpH7.0,usetheEquilibrationBuffer(pH8.0)insteadoftheEquilibration/WashBufferduringtheextractionandwashsteps.*Use250mMimidazoleinsteadof150mMimidazolewhenelutingfromTALONMagneticBeads.
Native purification of soluble polyhistidine-tagged protein
Denaturing purification of insoluble polyhistidine-tagged protein
Equilibrate resin
Elute
pH elutionBuffer at pH 5.0
Imidazoleelution
Buffer at pH 7.0 + 150 mM imidazole*
Imidazoleelution
Buffer at pH 7.0+ 150 mM imidazole*,
5.4 M guanidinium
Pure native protein Pure denatured protein
Wash nonadsorbedmaterial
Wash
Apply to TALON™ resin
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IV. Buffers for TALON® Purification and Buffer Kits
If you have purchased the TALON® Purification or Buffer Kits,preparebuf-fersasdescribedbelow.Todecreasetheamountofnonspecificallyboundproteins,werecommendusingtheEquilibration/Wash BufferatpH7.0duringpurification;however,ifyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbtotheresinatpH7.0,usetheEquilibrationBuffer(pH8.0)inplaceoftheEquilibration/WashBufferduringallextractionandwashsteps.NotethatatelevatedpHvalues,aminoacidsotherthanhistidine,aswellasthepeptidebond,canbeadsorbedbyTALONResins;Thus,underhighpHconditions(pH>8.0),proteinswithoutapolyhistidinetagcanbeadsorbed,decreasingresincapacityandthefinalpurityofyourtargetprotein.Note:Ifaprecipitateisobservedinthebuffers,warmthemat37°C,andstirorshaketodis-solveprecipitatepriortodilutingandusingthebuffers.
A. TALON xTractor Buffer: NopreparationnecessaryexceptoptionaladditionofDNaseIorLysozyme(seeSectionVII.A).
B. Equilibration Buffers 1.Diluteonepartofthe5XEquilibration/WashBufferor5XEquilibra-
tionBufferwithfourpartsofdeionizedwater. 2.CheckandcorrectpHifnecessary.The1XEquilibration/WashBuffer
shouldbepH7.0,whilethe1XEquilibrationBuffershouldbepH8.0.
C. Elution Buffer Diluteonepartofthe10XElutionBufferwithninepartsof1XEquilibra-
tion/WashBuffer(pH7.0)(or1XEquilibrationBuffer[pH8.0],dependingonthesolubilityofyourprotein)preparedinStepA.
D. Denaturing Conditions Add 6 M guanidinium to the Equilibration/Wash Buffer (pH 7.0), or
EquilibrationBuffer(pH8.0),andtheElutionBufferpreparedinStepsAandB,respectively.
Note: Performallstepsduringthepurificationprocedureinthepresenceof6Mguani-dinium.ProteinsamplescontaininghighguanidiniumconcentrationsformaprecipitatewhenloadedonSDSpolyacrylamidegels.Therefore,dialyzethesampleovernightinabufferedsolutioncontaining8Mureabeforeloadingitontothegel.
E. Wash Buffers • Ingeneral,usetheEquilibration/WashBufferatpH7.0towashnon-
adsorbedproteins.IftheproteinisnotstableatpH7.0,thenusetheEquilibrationBufferatpH8.0with5–10mMimidazole.
• Ifyourhostcell lineproducesunwantedmulti-histidineproteins,incorporateamorestringentwash:
Dilute10XElutionBufferineither1XEquilibration/WashBufferor1XEquilibrationBufferforafinalconcentrationof5–10mMimid-azole(1:300–1:150).
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If you have purchased the TALON® Magnetic Beads Buffer Kit (Cat.No.635638),preparebuffersasdescribedbelow. A. TALON xTractor Buffer: Nopreparationnecessaryexceptoptional
additionofDNaseIorLysozyme(seeSectionVII.A).
B. Equilibration Buffers 1.Diluteonepartofthe5XEquilibration/WashBufferwithfourparts
ofdeionizedwater. 2.CheckandcorrectpHifnecessary.The1XEquilibration/WashBuffer
shouldbepH7.0.
C. Elution Buffer 1.Dilute one part of the 4X Elution buffer with three parts of 1X
EquilibrationBuffer. 2.CheckandcorrectpHifnecessary.The1XElutionBuffershouldbe
pH7.0.
D. Wash Buffers • Ingeneral,usethe1XEquilibration/WashBufferatpH7.0towash
non-adsorbedproteins. • If your host cell line produces unwanted histidine-rich proteins,
incorporate a more stringent wash with 10 mM imidazole in 1XEquilibration/WashBuffer:
Dilute4XElutionBuffer(1Mimidazole)in1XEquilibration/WashBuf-ferforafinalconcentrationof5–10mMimidazole(1:200–1:100).
V. Buffers for TALON® Magnetic Beads
E. Denaturing Conditions Add6MguanidiniumtotheEquilibration/WashBuffer(pH7.0)andthe
ElutionBufferpreparedinStepsBandC,respectively. Note: Performallstepsduringthepurificationprocedureinthepresenceof6Mguani-
dinium.ProteinsamplescontaininghighguanidiniumconcentrationsformaprecipitatewhenloadedonSDSpolyacrylamidegels.Therefore,dialyzethesampleovernightinabufferedsolutioncontaining8Mureabeforeloadingitontothegel.
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A. Transformation of Host Cells with Expression Vectors Thefollowingprotocolisforchemically-inducedtransformationofE. coli competentcells.Performcontroltransformationsinparallel.
Note: UseJM109oranotherlac-induciblecelllinetoseeinductionofexpression.Fortightercontrolofexpressionlevels,useourPROTet6xHNBacterialExpressionSystem—especiallyrecommendedforexpressionofcytotoxicproteins.
1.Onice,thawatubecontaining100µlof0.5Mβ-mercaptoethanol(β-ME)andone50µltubeoffrozenE. colicompetentcellsforeachligation/transformation.
2.Dispense2µlof0.5Mβ-MEintoeachtubeofcompetentcellsandmix. 3.Dispense 2 µl of DNA directly into the mixture from Step 2. 4.Incubatetubesonicefor30min. 5.Heatshockforexactly30secina42°Cwaterbath. 6.Removetubesfromwaterbathandplaceonicefor2min. 7.Add250µlofSOCmediumtoeach tubeat roomtemperature. 8.Shake the tubes horizontally at 37°C for 1 hr at 225 rpm in
arotaryshakingincubator. 9.Spread transformation mixtures onto LB-ampicillin (50 µg/ml)
agarplates[containingX-gal(75µg/ml)andIPTG(1mM)ifblue-whiteselectionisdesired].Incubatetheplatesat37°Covernight.
B. Protein Expression 1.GrowanovernightcultureofE. colitransformedwithyourexpres-
sionplasmid.Ifyoucanisolateasufficientamountofproteinfromthisculture,proceedtoStep3aftertakinga1mlsampleforelectro-phoreticanalysis.Centrifugethesampleat1,000–3,000xgfor15minat4°C,removethesupernatant,andstorethecellpelletat–20°C.Note:Ifalarge-scalepreparationoftheproteinisrequired,proceedtoStep2.
2.Ifyouneedagreaterquantityofthetargetprotein,use20mlofovernightculturetoinoculate1Lofmedium.Incubatewithshakingforanother1–2hr,untiltheculturehasanabsorbanceof~0.6OD600.Removea1mlsampleoftheculture,centrifugeat1,000–3,000xgfor15minat4°C,removethesupernatantandstorethecellpelletat–20°Cforelectrophoreticanalysis.
3.Induceexpressionbyaddinganappropriateinducer.Forexample,thelacpromoterinthepHAT10expressionvectorcanbeinducedwith1mMIPTG.Continuetheincubationforanother3–5hr.
4.Removea1mlsampleoftheculture,centrifugeat1,000–3,000xgfor15minat4°C,removethesupernatant,andstorethecellpelletat–20°Cforelectrophoreticanalysis.
5.Proceedwithsamplepreparation(SectionVII).
VI. Transformation & Protein Expression
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VII. Sample Preparation
A. TALON® xTractor Buffer Sample Preparation
ThisprocedurehasbeenoptimizedforextractionofnativeproteinsfromfreshorfrozenbacterialcellpelletusingTALONxTractorBuffer(Cat.No.635623).Thevolumesofthisextractioncanbeadjusted,aslongas20mlofxTractorBufferareusedper1gofcellpellet.
1.Add20mlofxTractorBufferto1gofbacterialcellpellet.Mixgently.Pipetthemixtureupanddowntofullyresuspendthepellet.Notes
•ForTALONHT96-WellPlate(Cat.No.635622),resuspend40–100mgofbacterialcellpelletin2mlofxTractorBuffer.
•Alog-phasecultureofE. coli(O.D.=0.6–0.8)wheninducedfor2–4hours,wouldbeexpectedtoprovide~20–40mgbacterialpelletfrom2mloftheculture.
2.[Optional]:Add40µlof1unit/µlDNaseIsolutionand200µlof50Xlysozymesolution.Notes
•DNaseIreducestheviscosityofthelysate,allowingformoreefficientremovalofcellulardebris.DNasecanbeusedwithoutlysozyme.However,ifyouareyouaretreatingcellswithlysozyme,thenyoumust treatcellswithDNaseIaswell.
•Lysozymehelpstofullydisruptbacterialwalls,andthusithasbeendemonstratedtobehighlybeneficialinextractionofhighmolecularweightproteins(>40KDa).However,lysozymeshouldbeomittedformammalianextractionproceduresaswellaswhenlysozymeinterfereswithyourprotein'sfunctionality.
3.Mixgently,pipettingupanddownseveraltimes. 4.Incubatewithgentleshakingfor10minatroomtemperature.(You
mayincubatethesolutionat4°C,ifdesired).Note: Attheendofthisincubationperiod,thereshouldbenovisibleparticles.Ifcellpellet fragmentsarepresent, resuspendthembypipettingsolutionupanddownandincubatingforadditional1–2min.
5.TheresultingcelllysatecannowbeapplieddirectlytoaTALONCellThruColumn,orthelysatesupernatantcanbeappliedtoanyotherTALONResincolumnaftercentrifugingat10,000–12,000xgfor20minat4°C.
B. Standard Sample Preparation to Isolate Native Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15
minat4°C.Removethesupernatant.Ifyieldislow,usethemildextractionmethoddescribedinStep6,below.
2.Resuspendthecellpelletbyvortexingin2mlofchilled1XEquilibra-tion/WashBuffer(4°C)per25mlofculture≤100ml.Forcultures>1L,resuspendthepelletin1–2%oftheoriginalculturevolume.Note: You may omit Steps 3–4 if lysozyme treatment interferes with your protein’s functionality.
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VII. Sample Preparation continued
3.Addlysozymetothe1XEquilibration/WashBufferforaconcentra-tionof0.75mg/ml.Toreducethechanceofintroducingproteases,usethehighestpuritylysozymeavailable.
4.Incubateatroomtemperaturefor20–30min.Note: Incubations at room temperature result in elevated proteolytic activities.Alternatively,youcanuselysozymeat4°Cwithlowerefficiency.Ifthistreatmenthydrolyzesthetargetprotein,usethemethoddescribedinStep6(below).Alter-natively,disruptthecellsbyrepeatedfreeze/thawcycles;thatis,flash-freezingthecellsuspensioninadryice-ethanolbathandthawinginchilledH2O.
5.Ifyoursampleis≤50ml,sonicateit3x10sec,witha30secpauseonicebetweeneachburst.Ifyoursampleis≥200ml,sonicateit3x30sec,witha2minpauseonicebetweeneachburst.ProceedtoStep7.Note:Excessivesonicationcandestroyproteinfunctionality.
6.[Optional]: High-yield, mild extraction method.Transferthecellstoachilledmortarandgrind1partcellswith2.5partsalumina(SigmaCat.No.A-2039)for2–3minoruntilthecompositionofthemixturebecomespaste-like.Add2mlchilled1XEquilibration/WashBuffer(4°C)per25mlculture.Note:Ifthereisahighlevelofproteolyticactivityinthecelllysate,werecommendadding1mMEDTA(finalconcentration)totheEquilibration/WashBufferinordertoinhibitmetalloproteasesduringtheextraction.BeforeapplicationofthesampletotheTALONResin,EDTAmustberemovedbygelfiltrationchromatography(PD-10,GEHealthcare)equilibratedwiththeEquilibration/WashBufferforIMAC.
7.Centrifugethecellextractat10,000–12,000xgfor20minat4°Ctopelletanyinsolublematerial.
8.Carefullytransferthesupernatanttoacleantubewithoutdisturb-ingthepellet.Thisistheclarifiedsample.
9.Reserveasmallportionoftheclarifiedsampleat4°CforSDS/PAGEanalysis.
10.Ifthisisthefirsttimeyouhavepreparedclarifiedsamplesfromcellsexpressingaparticularrecombinantprotein,werecommendthatyouestimatetheprotein’sexpressionlevelinthathoststrain.Todoso,performasmall-scalepurification,andthenanalyzeaportion by SDS/PAGE in parallel with protein standards. Onceexpressionisobserved,proceedwiththeappropriatepurificationprotocol(seeSectionVIII).
C. Standard Sample Preparation to Isolate Denatured Proteins 1.Harvest20–25mlofcellculturebycentrifugationat1,000–3,000
xgfor15minat4°C. 2.Resuspendthepelletin2mlofdenaturing 1XEquilibration/Wash
Buffer(pH7.0)per20–25mlofculture. 3.Gentlyagitateorrthesampleuntilitbecomestranslucent. 4.Centrifugethesampleat10,000–12,000xgfor20minat4°Cto
pelletanyinsolublematerial.
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5.Carefullytransferthesupernatanttoacleantubewithoutdisturb-ingthepellet.Thisistheclarifiedsample.
6.Setasideasmallportionof theclarifiedsample forSDS/PAGEanalysis.Thenproceedwiththeappropriatepurificationprotocol(seeSectionVIII).Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.
D. Standard Sample Preparation for TALON® CellThru Resin
Sample Preparation to Isolate Native Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15
minat4°C.Removethesupernatant. 2.Resuspendthecellpelletbyvortexingin2mlofchilled1XEquilibra-
tion/WashBuffer(4°C)per25mlofculture≤100ml.Forcultures>1L,resuspendthepelletin1–2%oftheoriginalculturevolume.Note: You may omit Steps 3–4 if lysozyme treatment interferes with your protein’s function.
3.Addlysozymetothe1XEquilibration/WashBufferforaconcentra-tionof0.75mg/ml.Toreducethechanceofintroducingproteases,usethehighestpuritylysozymeavailable.
4.Incubateatroomtemperaturefor20–30min.Note: Incubations at room temperature result in elevated proteolytic activities.Alternatively,youcanuselysozymeat4°Cwithlowerefficiency.Ifthistreatmenthydrolyzesthetargetprotein,usethemethoddescribedinStep6.Alternatively,disruptthecellsbyrepeatedfreeze/thawcycles;thatis,flash-freezingthecellsus-pensioninadryice-ethanolbathandthawinginchilledH2O.
5.Ifyoursampleis≤50ml,sonicateit3x10sec,withapausefor30seconicebetweeneachburst.Ifyoursampleis≥200ml,soni-cateit3x30sec,witha2minpauseonicebetweeneachburst.Note:Excessivesonicationcandestroyproteinfunctionality.
6.Storeasmallportionoftheclarifiedsampleat4°CforSDS/PAGEanalysis.
Sample Preparation to Isolate Denatured Proteins 1.Harvest20–25mlofcellculturebycentrifugationat1,000–3,000
xgfor15minat4°C. 2.Resuspendthepelletin2mlofdenaturing 1XEquilibration/Wash
Buffer(pH7.0)per20–25mlofculture. 3.Gentlyagitateorstirthesampleuntilitbecomestranslucent. 4.Setasideasmallportionof theclarifiedsample forSDS/PAGE
analysis.Thenproceedwiththeappropriatepurificationprotocol,below(seeSectionVIII).Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.
VII. Sample Preparation continued
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E. Standard HT 96-Well Sample Preparation 1.Growcellsinappropriateformatforhigh-throughputanalysis. 2.Centrifugeifnecessaryandremovesupernatant. 3.Ifthetargetproteinsaresecretedinthemedium,utilizethesuper-
natantasastartingmaterialandproceedtoStep6.Ifthetargetproteinsareintracellular,proceedtothenextstep.
4.Disruptthecellsinpresenceof1XEquilibration/WashBuffer(use2mlofbufferper200mgofcellsperpurificationwell).Note:[Optional]UseTALONxTractorbufferforbetterextractionefficiency.
5.Centrifugeextractsandcollect thesupernatant tobeusedasastartingmaterial.
6.Remove50µlofeachsampleforproteinconcentrationanalyses.
F. Standard Sample Preparation for TALON Magnetic Beads When purifying proteins, more effective binding is achieved when
running clarified lysates. For screening of expression levels, crudelysatesderivedfromovernightculturescanbeaddeddirectlytothebeads(seeSectionVII.G).
Sample Preparation to Isolate Native Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15
minat4°C.Removethesupernatant.Thepelletcaneitherbeusedimmediatelyorstoredfrozenat–70°C.
2.Add0.5mlofxTractorBufferper25mgofcellpellet.ThevolumeofxTractorBuffercanbeincreasedordecreaseddependingonthesizeofthecellpellet.
3.[Optional]: Add1µlof1unit/mlDNaseIsolution. 4.Mixgently,pipetingupanddownseveraltimes. 5.Incubatewithgentleshakingfor10minatroomtemperature,or
at4°C,ifdesired. 6.[Optional]: High-yield, mild extraction method.Transferthecells
toachilledmortarandgrind1partcellswith2.5partsalumina(SigmaCat.No.A-2039)for2–3minoruntilthecompositionofthemixturebecomespaste-like.Add1mlchilled1XEquilibration/WashBuffer(4°C)per25mgofcellpellet.
7.Centrifugeat10,000-12,000xgfor20minat4°C.Note:TheuncentrifugedcrudecelllysatecanalsobeappliedtoTALONMagneticBeads.However,thelysatemayhavetobedilutedfurtherorrequiremoreDNasetopreventthebeadsfromfailingtomigratetothemagnetbecauseofthehighviscosityofthesolution.
8.Carefullytransferthesupernatanttoacleantubewithoutdisturb-ingthepellet.Thisistheclarifiedsample.
VII. Sample Preparation continued
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VII. Sample Preparation continued
9.Reserveasmallportionof theclarifiedsampleat4°CoroniceforproteinassaysandSDS-PAGEanalysis.ThenproceedwiththeTALONMagneticBeadspurificationprotocol(SectionVIII.I).
Sample Preparation to Isolate Denatured Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15
minat4°C.Removethesupernatant.Thepelletcaneitherbeusedimmediatelyorstoredfrozenat–70°C.
2.Add0.5mlofdenaturing 1XEquilibration/WashBufferper25mgofcellpellet.Thevolumeofthebuffercanbeincreasedordecreaseddependingonthesizeofthecellpellet.
3.Gentlyagitateorstirthesampleuntilitbecomestranslucent. 4.Centrifugethesampleat10,000–12,000xgfor20minat4°Cto
removeanyinsolublematerial. 4.Carefullytransferthesupernatanttoacleantubewithoutdisturb-
ingthepellet.Thisistheclarifiedsample. 5.Setasideasmallportionoftheclarifiedsampleat4°Coronice
forproteinassaysandSDS-PAGEanalysis.ThenproceedwiththeTALONMagneticBeadspurificationprotocol(SectionVIII.I).Note: Samplescontaining6Mguanidinemustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.
G. Sample Preparation Directly from Overnight Cultures for TALON Magnetic Beads
Ifscreeningtransformantsforexpressionlevels,pickasinglecolonyfromtheplateandinoculate4.5mlmedium.Incubatethecultureat37°CuntiltheOD600reaches~0.6–0.8AU(mid-logphase).Theninduceproteinexpressionwiththerecommendedconcentrationofinduceragent(dependingonyourexpressionstrainandtheexpressionplas-midbeingused).Continuetogrowtheculturewithrigorousshakingat37°Cforanother4hrorovernight.Alternatively,followyourstandardinductionorexpressionprotocol.
1.Diluteovernightculture1:1withxTractorBufferandaddDNasetoaconcentrationof1unit/mlofculture.(Forexample,dilute0.5mlofanovernightculturewith0.5mlofxTractorBufferandadd1unitofDNase.)
2.Mixthoroughlyat4°Cfor30min. 3.[Optional] Ifthecultureisstilltooviscous,diluteitwithsufficient
5X Equilibration/Wash Buffer to obtain a final concentration of1XEquilibration/WashBuffer.
4.CheckpHtoensureitfallsbetween7–8foroptimalbindingandproceed with theTALON Magnetic Beads purification protocol(SectionVIII.I).
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PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.
A. General Information 1.Performallmanipulationsat4–8°Cinordertomaintainprotein
stabilityandimproveyield. 2.This protocol is designed using the Equilibration/Wash Buffer
(pH 7.0).IfyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbatpH7.0,usetheEquilibrationBufferatpH8.0(insteadoftheEquilibration/WashBuffer)duringextractionandwashsteps.
3.Areducingagent,suchas10mMβ-ME,oraproteaseinhibitor,suchasPMSF,intheEquilibration/WashBuffer(pH7.0),mayimprovethestructuralstabilityoffragileproteinsduringsamplepreparation.SeeAppendixAforcompatibilityinformation.Note:Dependingontheconcentrationandvolumeoftheadditiveyouwishtouse,youmayneedtoremakethebufferstopreservetherecommendedconcentrationofNaClandbufferingagent. DTT and DTE are not compatible with this TALON protocol in any concentration.
4.If thecell lysatecontainsahighlevelofproteolyticactivity,werecommendadding1mMEDTAtotheEquilibration/WashBuffer(pH7.0)toinhibitmetalloproteasesduringtheextraction. However, before applying the sample to the resin, remove EDTA using a gel filtration column (such as PD-10, GE Healthcare) equilibrated with the Equilibration/Wash Buffer.Insomecases,thehostcellproduceslowmolecularweightchelatorsthatcanalsoberemovedusinggelfiltration.
Chelators can be detected by applying your sample to a smallcolumnpackedwithTALONResin.Ifthetopofthecolumnlosesitscharacteristicpinkcolor,andthecolorlessfrontmovesinthedirec-tionoftheflow,orifyouobtainpinkfractionsduringbatchadsorp-tion,youmustequilibratethesampleusingagelfiltrationcolumn.
5.Overexpressedrecombinantproteinscanaccumulateininsolubleinclusionbodies.Inordertodetermineoptimalextraction/purifica-tionconditions,youmustdeterminethedistributionoftheproteininsolubleandinsolubleforms.PerformapreliminarySDS/PAGEanalysis of protein extracts obtained under native conditions,followedbyextractionoftheresidualproteinsunderdenaturingconditions.Takecaretousethesameextractionvolumesforbothnativeanddenaturingextracts,andrunthecellextractbeforein-ductionasacontrolinonelanetoidentifythetargetprotein.Useofdenaturingconditions isrecommendedonly if thebiologicalactivityofthetargetproteinwouldnotbeaffectedbydenaturationorisunimportant.Itispreferabletousenativeconditionsforex-tractionevenifonly5–10%ofthetargetproteinissoluble.
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6.Thebuffervolumesinthefollowingprotocolswereoptimizedforpurifying theHAT-DHFR fusionprotein from20–25mlofE. coliculture.Dependingontheexpressionlevelandanticipatedyield,youmayneedtoadjustthebuffervolumesforotherproteins.Asastartingpoint,use2mlofbufferper20–25mlofculture.
7.Ifyouarepurifyingproteinfromharvestedeukaryoticcells,lysethecellsinanappropriatebuffercontainingamilddetergent(Sambrook&Russell,2001).SeeAppendixAforcompatiblebufferadditives.Note that EDTA and EGTA are not compatible with these protocols be-cause these metal-chelating reagents strip the cobalt from the resin.
8.Carefullycheckthesample’sappearanceafterlysisorsonication.BacterialsamplesoftenremainviscousfromincompleteshearingofgenomicDNA.CompleteDNAfragmentationimprovesproteinyieldsandallowsefficientremovalofcellulardebrisduringcentrifu-gation.Youmaydecreasethesample’sviscositybydigestionfor20–30minatroomtemperaturewith2.5µg/mlofDNaseI.Remem-berthatproteolyticactivityismuchhigheratroomtemperature.Alternatively,dilutethesamplefivefoldwithEquilibration/WashBufferbeforeapplyingittotheresin.Thisprocedureshouldnotsignificantlyaffectrecovery.
Notes on Protein Purification methods using TALON® Resin Thefollowinggeneralguidelinesareusedforpurifyingpolyhistidine-
taggedproteinfromtransformedE. colicultures.TableIprovidesanoverviewofTALON®Resinproteinpurificationmethodsandapplica-tions.Chooseamethodthatbestsuitsyourresearchneeds.
• Use2mlofresinsuspensionper~3mgofanticipatedpolyhisti-dine-tagged protein. 2 ml of homogeneously resuspended resinwillprovide1ml(bedvolume)ofTALONResin.
• Thebuffersandpurificationconditionsshouldworkwellformostsoluble,monomericproteinsexpressedinE. coli.
• Initially,testeachdifferentexpressionsystemandpolyhistidine-taggedproteininsmall-scalebatchpurificationtodetermineexpressionlevelsandtooptimizetheprotocol.Amini-scalebatchpurificationprotocolisprovidedinAppendixB;alternatively,youcanuseaTALONspincolumn.
• Purificationmethodsthatworkwithnickelorzinc-basedIMACresinsshouldalsoworkwiththeseresins.However,someoptimizationmayberequired.
TALON® CellThru Considerations Theprocedureforpurifyingpolyhistidine-taggedproteinsusingTALON
CellThruResinisessentiallythesameasotherTALONResinswiththefollowingsignificantdifferences.
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1. Extracellular Proteins Iftherearenochelatingagentsinthefermentationliquidandthe
pHis≥7.0,youcanapplysampledirectlyontoaprepackedcolumn.Otherwise, a desalting/equilibration step is necessary (such asultrafiltrationorgelfiltrationwithSephadexG25).
2. Intracellular Proteins Forpurifying intracellularproteins, apply the sonicatedsample
containingyourtargetproteins,directlyontoaprepackedcolumn.Thereisnoneedforcentrifugation.Electrophoresiswillrevealthatsomeofthetargetproteinhaspassedthroughthecolumnwithoutadsorption.Toalargeextentthematerialpassingthroughthecol-umnisinsolubleprotein,whichwouldnormallyhavebeenremovedduring high-speed centrifugation.The amount of non-adsorbedtargetproteinwillalsovaryasafunctionofsonicationefficiency.
3. Chromatography Considerations TALONCellThruBeadshaveadiameterof300–500µm;therefore,
useacolumnwithafilterporesizeof90–130µmtoadequatelypasscellulardebris.WerecommendusingourCellThru2ml&10mlDis-posableColumns(Cat.No.635512&635513).The2mlcolumnsaresuitablefor1–2mlbedvolumes,whilethe10mlcolumnsaresuitablefor5–10mlbedvolumes.Becausethecolumnfiltershavealargerporesizeandpermithigherflowrates,youmayneedtoincubateyoursamplewiththeresinfor5minbeforelettingitflowthrough.Ifnecessary,passthesamplethroughthecolumnasecondtime.
Thetechniqueofexpandedbedchromatographyworkswellwiththeseresinsasthematerialcanflowthroughtheresinmoreef-fectively.Flowratesmayhavetobeadjustedtogetthemaximumbindingefficiencywhenusingthistechnique.
B. Batch/Gravity-Flow Column Purification
For column IMAC usingTALON Resins, we recommend a hybridbatch/gravity-flowprocedure.Thismethodcombinesthespeedandconvenienceofabatchprocedurewiththeexceptionallyhighpurityofthegravity-flowcolumnmethod.Inthishybridprocedure,thebind-ingandinitialwashingstepsareperformedinabatchformattosavetime,eliminateextraneousdebris,andavoidcolumnclogging.Aftertheinitialwashes,theresinistransferredtoacolumnforadditionalwashingandproteinelution.
1.ThoroughlyresuspendtheTALONResin. 2.Immediatelytransfertherequiredamountofresinsuspensiontoa
steriletubethatwillaccommodate10–20timestheresinbedvolume. 3.Centrifugeat700xgfor2mintopellettheresin.
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4.Removeanddiscardthesupernatant. 5.Add10bedvolumesof1XEquilibration/WashBufferandmixbriefly
topre-equilibratetheresin. 6.Recentrifugeat700xgfor2mintopellettheresin.Discardthe
supernatant. 7.RepeatSteps5and6. 8.AddtheclarifiedsamplefromSectionVI.A,B,orCtotheresin. 9.Gentlyagitateatroomtemperaturefor20minonaplatformshaker
toallowthepolyhistidine-taggedproteintobindtheresin. 10.Centrifugeat700xgfor5min. 11.Carefullyremoveasmuchsupernatantaspossiblewithoutdisturb-
ingtheresinpellet. 12.Washtheresinbyadding10–20bedvolumesof1XEquilibration/
WashBuffer.Gentlyagitatethesuspensionatroomtemperaturefor10minonaplatformshakertopromotethoroughwashing.
13.Centrifugeat700xgfor5min. 14.Removeanddiscardthesupernatant. 15.RepeatSteps12–14. 16.Addonebedvolumeofthe1XEquilibration/WashBuffertothe
resin,andresuspendbyvortexing. 17.Transfertheresintoa2mlgravity-flowcolumnwithanend-cap
inplace,andallowtheresintosettleoutofsuspension. 18.Removetheend-cap,andallowthebuffertodrainuntilitreaches
thetopoftheresinbed,makingsurenoairbubblesaretrappedintheresinbed.
19.Washcolumnoncewith5bedvolumesof1XEquilibration/WashBuffer.
20.[Optional]:Ifnecessary,repeatStep19undermorestringentcon-ditionsusing5–10mMimidazolein1XEquilibration/WashBuffer(SectionIV.D).
21.Elutethepolyhistidine-taggedproteinbyadding5bedvolumesofElutionBuffertothecolumn.Collecttheeluatein500µlfractions.Note: Undermostconditions,themajorityofthepolyhistidine-taggedproteinwillberecoveredinthefirsttwobedvolumes.
22.UsespectrophotometricandSDS/PAGEanalysestodeterminewhichfraction(s)contain(s)thebulkofthepolyhistidine-taggedprotein.Note:UseaBradfordproteinassay(Bradford,1976)orUVabsorbanceat280nm.UseUVabsorbanceonlyifyouareelutingsufficientproteintoexceedtheabsorbanceoftheimidazoleat280nm.Alternatively,dialyzethefractionsovernightagainsttheEquilibration/WashBuffer,andthenmeasuretheirUVabsorbanceat280nm.
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C. Large-Scale Batch Purification Thismethodpurifiespolyhistidine-taggedproteinsfasterthangravity-
flowcolumns;however,batchwashesremoveimpuritieslessefficientlythangravity-flowcolumns.Therefore,theyrequirelargerwashbuffervolumestoobtainpurepolyhistidine-taggedproteins.
1.ThoroughlyresuspendTALONResin. 2.Transferrequiredamountofresintoaglassfilterwithaporesizeof
10–20µm. 3.Applyavacuumtothefiltertoremoveexcessethanol. 4.Add 5 bed volumes of deionized water to the resin, and apply
vacuum. 5.Add5bedvolumesof1XEquilibration/WashBuffertotheresin,
andapplyvacuum. 6.RepeatStep5twotimes. 7.Addcrudelysate(CellThruResin)orclarifiedsample(otherthan
CellThruResin)totheresin,andmixfor3–5min. 8.Applyvacuumandcollectthefiltrate. 9.Washtheresinbyadding10–20bedvolumesof1XEquilibration/
WashBuffer.Gentlyagitatethesuspensionatroomtemperaturefor10minonaplatformshakertopromotethoroughwashing.
10.Applyvacuumtoremovebuffer. 11.Repeattheabovewash(Steps9–10)2–3times. 12.[Optional]: If necessary, repeat Step 11 under more stringent
conditionsusing5mMimidazolein1XEquilibration/WashBuffer(SectionIV.D).
13.Elutethepolyhistidine-taggedproteinbyadding5bedvolumesofElutionBuffer.
14.Gentlyagitatesuspensionatroomtemperaturefor5min. 15.Apply vacuum, and collect the purified polyhistidine-tagged
protein. 16.RepeatSteps13–15twotimes,collectingseparatefractions. 17.Use spectrophotometric and SDS/PAGE analyses to determine
whichfraction(s)contain(s)themajorityofthepolyhistidine-taggedprotein.Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.
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D. Medium-Pressure Column Purification 1.Assemblecolumnaccordingtothemanufacturer’sinstructions. 2.ThoroughlyresuspendTALONSuperflowResin.Slowlypourthe
slurryintothecolumn,andavoidintroducingairbubbles. 3.Allowresintosettle.Acceleratethisprocessbyallowingthebuffer
toflowthroughthecolumnwithaperistalticpumpattachedtotheoutputofthecolumn.Donotexceedaflowrateof5ml/min/cm2.Donotallowtheresintodryout.Ifthisoccurs,resuspendtheresinandrepackthecolumn.
4.Insertandadjustthetopadaptorandconnectthecolumntothechromatographysystemaccordingtomanufacturer’sinstructions.Note:Avoidtrappingairbetweentheadaptorandtheresinsurface.
5.Equilibratethecolumnwith1XEquilibration/WashBuffer.Donotexceeda5ml/min/cm2flowrate.Monitortheeluantat280nm;thebaselineshouldbestableafterwashingwith5–10columnvolumes.
6.Applytheclarifiedsampletothecolumnafterfilteringitthrougha0.22-µmfilterandwashwithEquilibration/WashBufferuntilthebaseline(280nm)isstable.Monitorcolumnbackpressureduringsampleapplication.Startcollectingfractions.
Note:Ifthesampleisveryviscous,thecolumnpressuremayexceedtherecom-mendedvalue(150psi,1.0MPa).Reducetheflowrateordilutethesampletobringthepressureintoanacceptablerange.
Loadthesampleataflowrateof0.5–1.0ml/min/cm2toensurethatthepolyhistidine-taggedproteinwillbindtotheresin.Iftheproteindoesnotbind,reducetheflowratefurther.Ifdesired,increasetheflowrateforwashingandproteinelution.
Ifthetargetproteinisunstableatroomtemperature,performthechro-matographyat4°C.Alternatively,useflowratesupto5ml/min/cm2toload,wash,andelutetheprotein.Capacitywilldecreaseby10–15%,butonaverage,achromatographyrunshouldonlytake15–20min.
7.Washcolumnwith10–20column-volumesofEquilibration/WashBuffer,oruntilthebaselineat280nmisstable.Ifnecessary,add5–10mMimidazoletotheEquilibration/WashBuffer.
8.Elutethepolyhistidine-taggedproteinwith5–10column-volumesofElutionBuffer.Thepolyhistidine-taggedproteinusuallyelutesinthesecondandthirdcolumnvolumes.
9.UsespectrophotometricandSDS/PAGEanalysestodeterminewhichfraction(s)contain(s)themajorityofthepolyhistidine-taggedprotein.Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.
10.Ifyouplantostore,regenerate,andreusearesin-packedcolumn,seeSectionIX.C.
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E. 5 ml TALON® Single Step Column PurificationTheseprotocolsaredesignedforusewithTALONSingleStepColumnsforgravityfloworcentrifugepurification.• Forpurificationsoflessthan10samples,werecommendusingthe gravityflowprocedure.Forhigh-throughputpurificationofmorethan 10samples,thecentrifugeprocedureshouldbefaster.• Thebuffersusedinthisprocedurecanbeeasilypreparedbydilution
fromthestockbuffersfromourTALONBufferKit(Cat.No.635514).SeeSectionIVforpreparationdetails.
1.Samplepreparationandlysis
a.TALONSingleStepColumnscanbeusedforpurificationofanyhistidine-taggedproteinfromanE. coliculture.Forexample,ifscreeningtransformantsforexpressionlevels,pickasinglecolonyfromtheplateandinoculate4.5mlmedium.Incubatethecultureat37°CuntiltheOD600reaches~0.6–0.8AU(mid-logphase).Theninduceproteinexpressionwiththerecommendedconcentrationofinduceragent(dependingonyourexpressionstrain and the expression plasmid being used). Continue togrowtheculturewithrigorousshakingat37°Cforanother4hrorovernight.Alternatively, followyourstandardinductionorexpressionprotocol.[Optional:remove200µloftheexpressioncultureforSDS/PAGEanalysis.]
b.PlacethebottomclosurefirmlyonaTALONSingleStepCol-umn,removethetopcap,add4.5mlcultureandthenreplacethetopcap.Mixthesuspensioneitheronacarouselshakerfor20–30minatroomtemperatureorbyinvertingthetubeevery2minforatotalof30min.
c.Remove the top cap and the bottom closure and place theTALONSingleStepColumnintoaReceivingTube.Proceedwitheitherthegravityfloworthecentrifugeprocedure.
2.GravityFlowProcedure a.Lettheextractdrainbygravityflow.Removethecolumnfromthe
receivingtubeandreplacethebottomclosure.[Optional:remove200µlofnon-adsorbedmaterial fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis(Step4).]
b.Add4.5ml1XEquilibration/WashBuffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovethebottomclosureandputthecolumnintoaReceivingTube.Allowthewashtodrainbygravityflow.[Optional:remove200µlofWash-1fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice.
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d.Replace the bottom closure, then add 4.5 mlWash-2 Buffer.Replacethetopcapandresuspendtheresinbyinvertingthecolumn. Remove the bottom closure and put column into aReceivingTube.Letthebufferdrainbygravityflow. [Optional:remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
e.[Optional]Forimprovedpurityofthetargetprotein,repeatthewashinStepdtwice.
f.Add1.0mlElutionBufferandresuspendtheresinbyinvertingthecolumnsfor2min.
g.RemovethebottomclosureandputthecolumnintoaReceivingTube.Allowtheelutionfractiontodrainbygravityflow.ProceedwithStep4.ProteinAnalysis.
3.CentrifugeProcedure a.Centrifugeat700xgfor2min.TakethecolumnfromtheRe-
ceivingTubeandreplacethebottomclosure.[Optional:remove200µlofnon-adsorbedmaterialcollectedintheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
b.Add4.5ml1XEquilibration/WashBuffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovethebottomclosureandputthecolumnintoaReceivingTube.Centrifugeat700xgfor2min.RemovethecolumnfromtheReceivingTubeand replace thebottomclosure. [Optional: remove200µlofWash-1forSDS/PAGEandProteinAssayanalysis.]
c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice.
d.Add4.5mlWash-2Buffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovethebottomclosureandputtheSingleStepColumnintoaReceivingTube.Centrifugeat700xgfor2min.RemovethecolumnfromtheReceivingTubeand replace thebottomclosure. [Optional: remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAs-sayanalysis.]
e.[Optional]Forimprovedpurityofthetargetprotein,repeatthewashinStepdtwice.
f.Add1mlElutionBufferandresuspendtheresinbyinvertingthecolumnfor2min.
g.RemovethebottomclosureandputtheTALON®SingleStepColumnintoaReceivingTube.Centrifugethecolumninthetubeat700xgfor2min.ProceedwithStep4.ProteinAnalysis.
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4.ProteinAnalysis Determineamountofproteinina1:10dilutionofthenon-adsorbed
fractionsandtheamountofproteininthe(undiluted)elutionfractionbyperformingaBio-RadProteinAssay.AnalyzethesamplesbySDS/PAGEtodeterminethepurityofthetargetprotein(Elutionfraction).Note:ABCAProteinAssay (seeSection III) canbeperformedonanundilutedsampleofthenon-adsorbedfraction,ifdesired.
F. 20 ml TALON® Single Step Column PurificationTheseprotocolsaredesignedforusewiththe20mlTALONSingleStepColumnsforgravityfloworcentrifugepurification.• Forpurificationsoflessthan10samples,werecommendusingthegravityflowprocedure.Forhigh-throughputpurificationofmorethan10samples,thecentrifugeprocedureshouldbefaster.• Thebuffersusedinthisprocedurecanbeeasilypreparedbydilution
fromthestockbuffersfromourTALONBufferKit(Cat.No.635514).SeeSectionIVforpreparationdetails.
1.Samplepreparationandlysis
a.TALONSingleStepColumnscanbeusedforpurificationofanyhistidine-taggedproteinfromanE. coliculture.Forexample,ifscreeningtransformantsforexpressionlevels,pickasinglecolonyfromtheplateandinoculate25mlmedium. Incubatethecultureat37°CuntiltheOD600reaches~0.6–0.8AU(mid-logphase).Theninduceproteinexpressionwiththerecommendedconcentrationofinduceragent(dependingonyourexpressionstrain and the expression plasmid being used). Continue togrowtheculturewithrigorousshakingat37°Cforanother4–6hrorovernight.Alternatively,followyourstandardinductionorexpressionprotocol. [Optional: remove200µloftheexpressioncultureforSDS/PAGEanalysis.]
b.EnsurethattheendcapisfirmlyontheTALONSingleStepCol-umn,removethetopcap,add20mlcultureandthenreplacethetopcap.Mixthesuspensioneitheronacarouselshakerfor20–30minatroomtemperatureorbyinvertingthetubeevery2minforatotalof30min.
c.Remove the top cap and the end cap and place theTALONSingleStepColumnbackintotheReceivingTube.Proceedwitheitherthegravityfloworthecentrifugeprocedure.
2.GravityFlowProcedure a.Lettheextractdrainbygravityflow.Removethecolumnfrom
thereceivingtubeandreplacetheendcap.[Optional:remove200µlofnon-adsorbedmaterial fromtheReceivingTubefor
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SDS/PAGEandProteinAssayanalysis(Step4).] b.Add20ml1XEquilibration/WashBuffer,putthecolumninto
afreshReceivingTube,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovetheendcapandputthecolumnbackintotheReceivingTube.Allowthewashtodrainbygravityflow.[Optional:remove200µlofWash-1fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice. d.Replacetheendcap,putthecolumnintoafreshreceivingtube,
thenadd20mlWash-2Buffer.Replacethetopcapandresus-pendtheresinbyinvertingthecolumn.RemovetheendcapandreplacethecolumnintotheReceivingTube.Letthebufferdrainbygravityflow.[Optional:remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
e.[Optional] Forimprovedpurityofthetargetprotein,repeatthewashinStepdtwice.
f.Replacetheendcap,add2.0mlElutionBuffer,placethe20mlcolumnintoafreshreceivingtube,andresuspendtheresinbyinvertingthecolumnfor2min.Foranadditional10–15%ofpuri-fiedprotein,repeatelutionwithanadditional2.0mlElutionBuffer.
g.RemovetheendcapandreplacethecolumnintotheReceivingTube.Allowtheelutionfractiontodrainbygravityflow.ProceedwithStep4.ProteinAnalysis.
3.CentrifugeProcedure a.Centrifugethecolumnat700xgfor2min.Takethecolumnfrom
theReceivingTubeandreplacetheendcap.[Optional:remove200µlofnon-adsorbedmaterialcollectedintheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
b.Placethecolumninafreshreceivingtube,thenadd20mlof1XEquilibration/WashBuffer,replacethetopcapandresuspendthe resin by inverting the column. Remove the end cap andreplacethecolumnintotheReceivingTube.Centrifugeat700xgfor2min.[Optional:remove200µlofWash-1forSDS/PAGEandProteinAssayanalysis.]
c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice. d.RemovethecolumnfromtheReceivingTubeandreplacethe
endcap.Placethecolumninafreshreceivingtube,add20mlWash-2Buffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovetheendcapandreplacetheSingleStepColumnintotheReceivingTube.Centrifugeat700xgfor2min.[Optional:remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]
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e.[Optional]Forimprovedpurityofthetargetprotein,repeatthewashinStepdtwice.
f.RemovethecolumnfromtheReceivingTubeandreplacetheendcap.Add2mlElutionBuffer,placethecolumninafreshReceiv-ingtube,closethetopcapandresuspendtheresinbyinvert-ingthecolumnfor2min.Foranadditional10–15%ofpurifiedprotein,repeatelutionwithanadditional2.0mlElutionBuffer.
g.RemovetheendcapandreplacetheTALONSingleStepColumnintotheReceivingTube.Centrifugethecolumninthetubeat700xgfor2min.ProceedwithStep4.ProteinAnalysis.
4.ProteinAnalysis Determineamountofproteinina1:10dilutionofthenon-adsorbed
fractionsandtheamountofproteininthe(undiluted)elutionfractionbyperformingaBio-RadProteinAssay.AnalyzethesamplesbySDS/PAGEtodeterminethepurityofthetargetprotein(Elutionfraction).Note:ABCAProteinAssay (seeSection III) canbeperformedonanundilutedsampleofthenon-adsorbedfraction,ifdesired.
G. TALONspin™ Column Purification Important Points • Beforeproceedingwithpurification,determinetheconcentrationof
polyhistidine-taggedproteininyoursampleusingthemini-batchscreeningprotocol(AppendixB).Alternatively,runasampleoftheclarifiedlysatedirectlyonSDS/PAGE,andestimatetheamountofpolyhistidine-taggedproteinbybandintensity.
• Avoidexcessivelyconcentratedorviscouslysates. SeeTroubleshoot-ing(SectionIX.B.2)fortipsonreducingsampleviscosity.
• Iftheconcentrationofpolyhistidine-taggedproteininthelysateisverydilute,useonecolumntoenrichtheproteinfromseveral0.6–1mllysatealiquots.SimplyrepeatSteps11–16(below)untilthedesiredamountoflysatehasbeenprocessed.Alternatively,concentratethepolyhistidine-taggedproteinbyreducingthesamplevolume.
• Thecentrifugationrotorandspeedmayaffectyourresults.Ideally,youshouldcentrifugeTALONspinColumns inaswingingbucketrotor to allow the sample to pass through the resin uniformly.However,afixedanglerotororamicrocentrifugeisalsoacceptable.Centrifugationspeedshigherthan700xgmaycauseirregularitiesintheflowofsolutionthroughtheresinbed,andthus,decreasetheperformanceofthecolumn.
1.HoldtheTALONspinColumnuprightandflickituntilallresinfallstothebottomofthecolumn.Then,snapoffthebreakawayseal.Note:Savewhiteend-capforlateruse.
2.Placecolumninthe2mlmicrocentrifugetube.
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3.Removethecleartop-capandcentrifugecolumnat700xgfor2mintoremovethestoragebufferfromtheresinbed.Note:Theresinbedwillappearsemi-dryaftercentrifugation.
4.Removecolumnfromcentrifuge,andplacethewhiteend-capoverthemaleluerfitting.
5.Add 1 ml 1X Equilibration/Wash Buffer and mix briefly to pre-equilibratetheresin.
6.Recentrifugeat700xgfor2mintopellettheresin.Discardthesupernatant.
7.RepeatSteps7and8,twice. 8.AddtheclarifiedsamplefromSectionVII.A,BorCtotheresin. 9.Add0.6–1mlofsampletothecolumn,andreplacethecleartop-cap. 10.Allowsampletopassivelywettheresinbedfor30sec. 11.Mixorvortexcontentsbrisklyfor1–2sec,completelyresuspending
theresininthelysate. 14.Gentlyagitate thesuspension for5min toallowpolyhistidine-
taggedproteinbinding.Do not vortex. 15.Removebothcapsfromcolumnandplacecolumninsidethe2ml
microcentrifugetube. 16.Centrifugeat700xgfor2min. 17.Removethecolumnandmicrocentrifugetubefromthecentrifugerotor,
makingsurethatallofthesamplehaspassedthroughtheresinbed.Note:Viscoussamplesmayrequireadditionalcentrifugation.
18.Savethe2mltube,butdiscardtheflowthrough. 19.Placemicrocentrifugetubeinrotor. 20.Placewhiteend-caponthecolumn,andadd1mlof1XEquilibra-
tion/WashBuffer.Closethecolumnwiththecleartop-cap. 21.Allowthebuffertopassivelywettheresinbedfor30sec. 22.Agitateorvortexbrisklyforafewsecuntiltheresiniscompletely
resuspended. 23.Gentlyagitatefor5min. 24.Removebothcaps,andcentrifugeat700xgfor2min. 25.Repeat Steps 18–24. Repeat twice for particularly concentrated
lysates,orifnecessary,toimprovepurity. 26.Examinetheresinbedtoensurethatitappearssemi-dry,andto
ensurethatallwashbufferhasdrainedfromtheresinbedandthecolumnend.
27.Discardtheused2mlmicrocentrifugetube. 28.Ifnecessary,repeatthespintoremovealltracesofwashbuffer.
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VIII. Protein Purification Protocols continued
29.Replacethewhiteend-caponthespincolumn. 30.Add400–600µlofElutionBuffer.
Note: Alternatively,use100mMEDTA(pH8.0)ifitdoesnotinterferewithdownstreamapplicationsoftheprotein.SampleselutedwithEDTAwillalsocontaincobalt.
31.Allow1minforElutionBuffertopassivelywettheresinbed. 32.Brieflyagitateorvortextoresuspendtheresin. 33.Placeafresh2mlcollectiontubeintocentrifugerotor. 34.Removebothcapsandplacecolumnintothe2mlcollectiontube. 35.Centrifugesampleat700xgfor2min. 36.RepeatSteps30–35.
Note:The polyhistidine-tagged protein sample can generally be recovered in800–1,200µlofElutionBuffer,butitmaybenecessarytousealargerElutionBuffervolumeorrepeatSteps30–35.
37.Determinepolyhistidine-taggedproteinyieldusinggelorspectro-photometricanalysis.Note:Ifthepurityofthepolyhistidine-taggedproteinpreparationisunsatisfactory,refertotheprocedureintheTroubleshootingGuideSectionX.C.2.
H. TALON® HT 96-Well Purification ProtocolEachwelloftheTALONHT96-WellPlatehasacapacityofupto1.0mgofpolyhistidinetaggedprotein.Inordertoobtainthemaximumyieldofpureprotein,donotattempttoloadmorethan1.0mgofpolyhisti-dine-taggedprotein/well.Also,observethefollowingguidelines:
•Whenusingpipettetipstomixtheresin,usewide-boretips,orcutthe tips tomake theopeningwider.Thiswill reducemechanicaldamagetoproteinsaswellasresin.
•TALONResinisdesignedtopermitbuffertoflowthroughfreely.Therefore,whentheHT96-WellPlateisnotonthevacuummanifoldoroveraCollectionPlate,werecommendthatitiskeptontheBaseSealthatactsasatemporarystopper.
•Theamountofsampleappliedtoawellshouldnotexceedtheca-pacityof1.0mg/well.
•Avoidoverdryingtheresinunderthevacuum.Forthebestresults,keeptheresinwet.
•Whenusingvacuummanifold,adjustthevacuumtoobtainaflowrateof1–2dropspersec(~100–200mmHgor2–4psi).
1.Unpackingandremovalofseals HT96-Wellplatescomewithsolidplatesealstopreventresinfrom
leakingduringtransportation.Beforeremovingtheupperseal,werecommendperforminga2mincentrifugationstepat500xgtopack resinthatmighthaveadheredtothesiliconlidduringtransportation.
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Afterthisprocedure,theuppersealcanberemovedandthestepsoutlinedinthepurificationprotocolscanbeperformed.
Ifyoudonotdesiretouseall96wells,theplatesealscanbecutsothatonlythewellsthatareneededareexposed.Afterchromatogra-phy,theremovedportionoftheplatesealscanbereplacedandtheplatecanbestoredat4°Cuntiltheremainingwellsareused.Whenstored,theresininunusedwellsshouldbecoveredin20%ethanol.
2.HT96-WellPlateEquilibration a.Removethetopandbottomsealsfromtheplate. b.Placetheplateonthemanifoldandapplyvacuumtoremove
storagesolutionorcentrifuge5minat700xg. c.Add1mlofdeionizedwatertoeachwelloftheplate.Applyvacuum
orcentrifugetodrainthewaterfromthewells.Repeattwice. d.Add1mlof1XEquilibration/WashBuffertoeachwelloftheplate
andapplyvacuumorcentrifuge todrain thebuffer fromthewell.Repeattwice.
3.VacuumPurification Whenperformingvacuumpurification,adjustthevacuumtoobtain
aflowrateof1–2dropspersec(~100–200mmHgor2–4psi).Inaddition,avoidoverdryingtheresinwhichintroducesairbubblesandreducesperformance.
a.Apply1.5mlofthestartingsample(SeeSectionVII.E)perwell.Mixthesamplewiththeresinshortlybyvortexingtheplateorpipettingupanddowninsidethewells.Leavetheplateonicefor5–10minmixingsamplesevery2min.
b.Placetheplateonthevacuummanifold,applyvacuumandlettheexcessliquiddrainintothemanifold.Firmlypressallfoursidesoftheplatetotherubbergasketofthevacuummanifold.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
c.RepeatSteps3.aand3.bifadditionalloadingisnecessary. d.Add1mlof1XEquilibration/WashBufferandsuspendtheresin
byvortexingtheplateorpipettingupanddowninsidethewells. e.Placetheplateonthevacuummanifold,applyvacuumandlet
theexcessliquiddrainintothemanifold.Firmlypressallfoursidesoftheplatetotherubbergasketofthevacuummanifold.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
f.RepeatSteps3.dand3.etwice. g.Add1mlofHT96-WellPlateWashBuffer(SeeSectionIII)toeach
wellandsuspendtheresinbyvortexingtheplateorpipettingupanddowninsidethewells.
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h.Placetheplateonthevacuummanifold,applyvacuumandlettheexcessliquiddrainintothemanifold.Firmlypressallfoursidesoftheplatetotherubbergasketofthevacuummanifold.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
i.RepeatSteps3.gand3.hfivetimes. j.RemovetheHT96-WellPlatefromthevacuummanifold.Drain
thecollectedfiltratefromthevacuummanifold. k.PlaceaCollectionPlateinsidethevacuummanifoldandplace
theHT96-WellPlateonthevacuummanifold. Note:Beforeeluting,placetheplateoveraCollectionPlateoronthebaseseal.
l.Add200µlof1XElutionbuffer(SectionIII.A)andsuspendtheresinbyvortexingtheplateorpipettingupanddowninsidethewells.
m.Place the HT 96-Well Plate on the vacuum manifold, applyvacuum,andlettheeluatedrainintotheCollectionPlate.
n.Repeatelution(Steps3.mand3.n)twice. o.Determineamountofloadedandadsorbedproteinineachwell
byBradfordAssay(Bradford,1976).
4.CentrifugePurification Asavarietyofrotorsandcentrifugescanbeused,thefollowing
instructionsareonlygeneralguidelinesforsuccessfulpurification: • Donotutilizecentrifugalforcehigherthan700xg. • EnsureproperbalanceoftheHT96-WellPlate/CollectionPlate
insidetherotor. • Whenperformingthecentrifugeprocedurebelow,extraCollec-
tionPlatesarerecommended.SeeAdditionalMaterialsRequiredforinformationonobtainingcompatibleplates.
a.Add1.5mlofthestartingsampleperwell(SeeSectionVII.E).Mixthesamplewiththeresinbrieflybyvortexingtheplateorpipettingupanddowninsidethewells.Leavetheplateonicefor5–10minmixingsamplesevery2min.
b.Centrifugetheplatefor5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
c.RepeatSteps4.aand4.bifadditionalloadingisnecessary. d.Add1mlof1XEquilibration/WashBufferandsuspendtheresin
byvortexingtheplateorpipettingupanddowninsidethewells. e.Centrifugetheplatefor5min.Ensurebyobservationthatall
wellshavebeendrainedofbuffer. f.RepeatSteps4.dand4.etwice. g.Add1mlofHT96-WellPlateWashbufferandsuspendtheresinby
vortexingtheplateorpipettingupanddowninsidethewells.
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h.CentrifugetheHT96-WellPlatefor5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
i.RepeatSteps4.gand4.hfivetimes. j.Drain thecollectedfiltrate fromtheCollectionPlateorusea
freshCollectionPlate(SeeSectionIII.E). k.PlacetheHT96-WellPlateontheCollectionPlateintherotor
andcentrifuge5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
Note:Beforeeluting,ensurethattheplateisoveraCollectionPlateoronthebaseseal.
l.Add200µlof1XElutionbuffer(SectionIII.A)andsuspendtheresinbyvortexingtheplateorpipettingupanddowninsidethewells.
m.CentrifugetheHT96-WellPlateontheCollectionPlatefor5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.
n.Repeatelution(Steps4.land4.m)twice. o.Determineamountofloadedandadsorbedproteinineachwell
byBradfordAssay(Bradford,1976).
I. TALON® Magnetic Beads Purification ProtocolThisprotocolprovidesinstructionsforcarryingouttheTALONMagneticBeadspurificationinasingletube.ThebuffersusedinthisprocedureareeasilypreparedbydilutionfromthestockbuffersinourTALONMagneticBeadsBufferKit(Cat.No.635638).SeeSectionVforgeneralbufferpreparationguidelines.
1.Bufferpreparation a.Prepare5mlof1XEquilibration/WashBuffer • Ifthe5Xstockbufferisprecipitated,placebottleat37°C
for5min,thenshakeuntiltheprecipitatedissolves. • Dilute1mlof5Xstockwith4mlofH2O,confirmthatfinal
pHis7.0andcorrectpHifnecessary. b. Prepare0.5mlofElutionBuffer Add0.125mlof4XElutionBufferto0.375mlof1XEquilibra-
tion/WashbufferandconfirmthatthefinalpHis7.0.Anyunuseddilutedbuffercanbestoredandusedlater.
2.Generalconsiderationsforworkingwithmagneticbeads a.Useapipettetomixbufferthoroughlywiththebeads. b.Ifneeded,magneticbeadscanbemixedusingavortexer. c.Ifthereisagreatdealofliquid/bufferadheringtothesidesofthe
tube,centrifugethetubesinamicrofugebeforeplacingthemonamagneticseparator.
d.Ensurethatthebeadsareadheringtothesidesofthemagnetbeforeremovingthesupernatant.
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VIII. Protein Purification Protocols continued
3.Proteinpurificationundernativeordenaturingconditions a.Aliquot100–200µlofbeadsintoa1.5mlmicrofugetube. b. Placethetubeonamagneticseparatorfor1minandremove
storagebuffer. c. Add0.5mlofdeionizedwatertothebeads. d. Mixtheliquidandthebeadsthoroughlyusingapipette. e. Placethetubeonamagneticseparatorandremovethesuper-
natant. f. Toequilibratethebeads,add0.5mlof1XEquilibration/Wash
Buffer. g. Repeatstepsdande. h. Addthecelllysate(fromSectionsVII.ForVII.G)tothebeads. Note:Ifthecelllysatevolumeislessthan200µl,addsufficient1XEquilibra-
tion/WashBuffertobringthevolumeuptoatleast200µl.Thisisnecessarytoensurethoroughmixingofbeadswiththecelllysate,foroptimalbinding.
i. Mixonarotaryshakerfor30minatroomtemperature. Note:Iftheproteinisvulnerabletodegradationatroomtemperature,incubate
at4°Cfor1hr.ProteaseinhibitorsthatdonotcontainEDTAcanalsobeaddedduringtheincubation.
j. Placeonamagneticseparatorandcollectthesupernatant. k. Add0.5mlof1XEquilibration/WashBuffer. l. Mixthoroughlyandletitstandfor1minbeforeplacingona
magneticseparatorandcollectingthefirstwash. m. Repeatstepskandltwicetocollectthesecondandthirdwashes,
respectively. n. [Optional]: If necessary, repeat steps k and l under more
stringent conditions using 0.5 ml of 5–10 mM imidazole in1XEquilibration/WashBuffer(sectionV.D)
o. Toelutetheprotein,add50µlofElutionBuffer.ThevolumeofElutionBuffercanbevarieddependingontheamountofbeads used. 50 µl of elution buffer can be used for elutingfrom200µlofbeadsuspension.Mostoftheproteinwilleluteinthisfraction.Smallervolumes,suchas25µlcanbeusedifaconcentratedsampleisneeded.Volumesbelow25µlmaybedifficulttohandle.
p. Mixfor5minandcollectEluate1. q. Addanother50µlofElutionBuffer r. Mixfor1minandcollectEluate2.
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VIII. Protein Purification Protocols continued
s.Ifnecessary,stepsqandrcanberepeatedtwicetoensurethatproteinrecoveryismaximized.Inaspecificinstance,whenusing200µlofbeadsuspension,60%ofthetotalproteinwaselutedinthefirst50µlfraction,20%inthesecond,10%inthethird&5%inthefourth.
t. UsespectrophotometricandSDS-PAGEanalysestodeterminewhichfractionscontainthebulkofthepolyhistidine-taggedprotein.
Note:ABradfordproteinassayisrecommendedformeasuringproteinyields.Since thedetergents in thexTractorBuffermay interferewith theBradfordassay,itisadvisabletoruntheoriginallysateandnon-adsorbedfractionata1:5dilutionoruseaBCAassayforundilutedsamples.
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IX. Resin Washing, Reuse, and Storage
Generally,reuseTALON®Resins3–4timesbeforediscardingorcompleteregeneration.Theexactnumberofusesvariesamongpreparationsandapplicationbecauseofdifferencesinredoxpotential,organiccomplexity,anddebriscontent.Toavoidpossiblecross-contamination,useaparticularaliquotofresintopurifyasingletypeofpolyhistidine-taggedprotein.
Important precautions• TALONspin™Columnsarenotreusable.• DonotstoreTALONResinindenaturantssuchas6Mguanidinium.• DonotstoreTALONResinwithbound imidazole: the resinshouldbe
washedwithMESBuffer(pH5.0)describedinSectionIII,whichisrequiredbeforereusetoremovetheboundimidazole.
A. Stringent Wash (optional) 1.Washresinwithfourbedvolumesof6Mguanidinium(pH5.0)+
1%nonionicdetergent. 2.RinseresinwithfivebedvolumesofdistilledH2O. 3.Storeresinat4°Cin20%nonbufferedethanolcontaining0.1%azide.
B. Removing Imidazole 1.Washresinwithfivebedvolumesof20mMMESBuffer(pH5.0)
containing0.1MNaCl. 2.RinseresinwithfivebedvolumesofdistilledH2O. 3.Storeresinat4°Cin20%nonbufferedethanolcontaining0.1%azide.
C. Regeneration of Superflow Columns Purificationofpolyhistidine-taggedproteinsusingimidazolegradients
willcausethecolumntotakeonapurplishhue.Washingthecolumnwith5–10columnvolumesof20mMMESBuffer(pH5.0)willrestorethenormalpinkcolorandbringtheabsorbanceat280nmback to theoriginalbaselinelevel.AfterequilibratingthecolumnwithEquilibra-tion/WashBuffer,thecolumnisreadyforreuse.
D. Complete Regeneration 1.Striptheresinofcobaltionsbywashingwith10bedvolumesof
0.2MEDTA(pH7.0). 2.WashexcessEDTAfromtheresinwithanadditional10bedvolumes
ofdoubledistilledH2O(ddH2O). 3.Chargetheresinwith50mMCoCl2solution(10bedvolumes). 4.Washresinwith7bedvolumesofddH2Ofollowedby3bedvolumes
of300mMNaCl,and3bedvolumesofddH2Otoremoveexcesscobaltmetalions.
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5.EquilibratetheresinwithEquilibration/Washbuffer(10bedvolumes). 6.Ifyouplanttouseβ-mercaptoethanolinsubsequentbuffers/pro-
cedures,thenre-equilibratetheresinasfollowsbeforeproceedingwithfutherpurifications:
a.WashtheresinwithatleasttwobedvolumesofEquilibration/WashBuffer.
b.Re-equilibratetheresinwithEquilibration/WashBuffercontainingβ-mercaptoethanol.
E. TALON Magnetic Beads TALON Magnetic Beads are for single use only.They cannot be
regenerated.
IX. Resin Washing, Reuse, and Storage continued
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X. Troubleshooting Guide
A. Protein Expression
1.Noexpression
• Bad vector construct Checksequenceofthevector.
• Bad transformation Makeaplasmidminiprepandconfirmsequence.
• No inducing agent added to culture to induce expression
2.Apparentlowexpression
• Insoluble overexpressed protein Usedenaturingextractionandpurificationconditionsorreduce
expressionlevelsbyloweringtheamountofinducer.
• Unsuitable expression conditions Checkcellgrowthandinducerconcentration;checkforwild-type
(nontransformed)orantibioticresistantcells.
• Protein is secreted UsefermentationliquidasstartingsampleforIMACafterproper
buffering.
B. Loading/Washing
1.Polyhistidine-taggedproteinelutesinthewashbuffer
• Problems with vector construction Ensurethatproteinandtagareinframe.
• Buffer is not optimal CheckthepHandcompositionofallbuffers.Usealowerstringency
washbufferforallwashingsteps.Forexample,slightlyincreasethepHofthewashbufferorloweritsimidazoleconcentration.
• Protein degraded during extraction a.Usemildextractionconditions in thepresenceofprotease
inhibitors (e.g.,β-MEandEDTA)at4°C. Be sure to remove EDTA before applying to TALON®Resin.
b.MakeC-terminalconstruct.
c.Workquicklyat4°Ctoreducethetimeforinitialpurificationsteps.
• Reagent interferes with binding SeeAppendixAforreagentcompatibilities.Diluteanaliquotof
lysate(1:10),orsonicate,andcheckbindingonasmallscale.Tryusingadifferentpolyhistidine-taggedproteinasacontrol.
• Tag is not accessible under native conditions Iftheproteinfailstobindundernativeconditions,treatasmall
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aliquot(<1ml)with6Mguanidiniumandbindto50µlofresin.Thenfollowthemini-scaleprocedureinAppendixB.Ifthetargetproteinbindstotheresinunderthedenaturingconditions,thentrytomovethetagtotheotherterminusoftheproteinwhereitmaybemoreexposed.
2.Highbackpressureduringloadofsample
• High viscosity due to presence of DNA UseDNaseIordilutesamplefivefold(SectionVIII.A.8).
C. Elution
1.Highamountofco-elutedimpurities
• Insufficient wash UselargervolumesofEquilibration/WashBuffer.
• Buffer compositions are not optimal a.Checkbuffersusedforsamplepreparationandwashsteps. b.Check pH.The Equilibration/Wash Buffer should be pH 7.0.
Contaminantswillco-eluteinbuffers<pH7.0. c. Increasevolumeofwashbufferandcontinuetowashresin
beduntiltheA280dropstozero. d.Increasecounterionconcentrationupto0.5MNaClorKClto
inhibitnonspecificionicinteractions. e.Addethyleneglycolorglyceroltoinhibitnonspecifichydro-
phobicinteractions. f. Addsmallamountsofnonionicdetergent(s);thisisparticularly
importantwhenisolatingproteinsfromaeukaryoticexpres-sionsystem.
g.Add5–10mMimidazoletotheEquilibration/WashBufferanduseitasanintermediatewashstepbeforeelution.
• Proteolytic product Usemildextractionconditionsinpresenceofproteaseinhibitors
(e.g.,β-MEandEDTA)at4°C. Remove EDTA before applying to TALON®Resin.Proteinscanbeextractedinpresenceofproteaseinhibitorsspeciallydesignedforpurificationofhistidine-taggedproteinsastheydonotcontainEDTA.
• Covalent attachment (Cys-Cys) of impurities to the protein Use5–10mMofβ-MEintheEquilibration/WashofBuffer.
• Co-purifying histidine rich proteins a.ForHATproteins,useenterokinasetoremoveHATtagand
rerunIMACwithmixture.Targetproteinwillpassthroughthecolumn,whileimpuritiesandtagwillbeadsorbed.
X. Troubleshooting Guide continued
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Note:Removechelatingligandsbygelfiltrationbeforeloadingtheproteolytic mixtureontoTALONResin.
b.Usesecondpurificationprinciple,suchassizeexclusion,ionexchange,hydrophobic,orthiophilicchromatography.
• Protein sample is too concentrated and/or viscous Dilutesample1:5or1:10withadditionalbufferandcentrifuge
againbeforeproceeding.Also,seethenoteonreducingsampleviscosityaftersonicationinSectionVII.A.8.
2.ExcessivebackgroundafterTALONspin™Columnprocedure
• Sample is too viscous a.Reducetheviscosityofthesample(SectionVIII.A.8). b.DiluteclarifiedsamplewithanequalvolumeofEquilibration/
WashBufferandloadastwoaliquots. c. Increasenumberof1mlwashes. d.UseEquilibration/WashBuffer(pH7.0). e.Add1–5mMimidazoletoEquilibrationBuffer,pH8.0anduse
itasanintermediatewashstepbeforeelution. f. Tore-purifyasample,performthefollowingafterStep37in
SectionVIII.G: 1.Add4volumesofEquilibration/WashBuffertosemi-purifiedsample. 2.LoadsampleontoanotherTALONspin™Column. 3.Washtwicewith1mlofEquilibration/WashBuffer.
4.Eluteasbefore(StepsVIII.G.30–35).
3.Columnceasestoflow
• Filter is clogged with subcellular debris Changecolumnfiltersandcentrifugesampleat12,000xgfor
20–30minat4°C.
• Proteins precipitated on the column Use a mild detergent such as Decanoyl-N-methylglucamide
(MEGA-10, Sigma Cat. No. D6277) in the Equilibration/WashBuffer.
• The lower resin bed support may be clogged with cellular debris a.Removeresinfromcloggedcolumnandresuspend.Thenwash
itinabatchformatandtransfertoafreshcolumn. b.Useasyringefilledwithwashbufferorreversethepumpon
thecolumntogentlyrunthecolumnbackwards.Inaddition,test for tubingblockages inasimilarmanner.Applygentlepressure.Donotexceeda1drop/secflowrate.
X. Troubleshooting Guide continued
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4.Polyhistidine-taggedproteinsdonotelute
• Elution Buffer is less than optimal
a.Elutewith150mMimidazoleorpH4.0buffer.
b.Forreallytoughelutionproblems,youcanstripofftheproteinusing100mMEDTA(pH8.0);however,doingsowillremovethecobaltfromtheresinanddeposititinyourproteinsample.
c. Add1–5mMβ-MEtoreducedisulfidelinkages.Supplementbufferwith1%nonionicdetergent.
d.Purifypolyhistidine-taggedproteinunderdenaturingconditions.
D. Changes in Resin
1.Resinchangesfrompinktowhite—LossofCo2+
• Presence of chelators in sample Removechelatorsfromsamplebygelfiltration RegenerateresinasdescribedinSectionIX.D.
2.Grayorbrownresin
• TALON® Resin exposed to reducing agents or high concentration of β-ME
Completelyremovereducingagents,suchasDTEorDTT,orifpossible,bygelfiltrationwithβ-ME.Reduceβ-MEconcentration(≤5mM).
3.Resinparticlesaggregateorexhibitchangeinconsistency
• DNA cross-linking a.Increaseionicstrengthofthebuffersbyusing
≤500mMNaClorKCl.
b.VigorouslysonicatesampletoshearDNA.
c. Pretreatsamplewith100µg/mlDNaseIat30°Cfor30min.
d.Dilutesample1:5–1:10withbuffer,andrepeat.
e.Avoidlong-termstorageindenaturants.
E. Analysis
1.Highbackgroundonsilver-stainedgels
• Nucleic acid a.Supplementbufferwith0.2–0.5MNaClorKCl.
Repeatpurification.
b.ShearDNAmorevigorously.
c. UseDNaseIintheextractionprocedure.
X. Troubleshooting Guide continued
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X. Troubleshooting Guide continued
2.Nonfunctionalprotein
• Protein was damaged by sonication a.Conduct a time-course assay to determine the minimum
sonicationtimeneededtodisruptthecellswhilemaintainingthe native protein/enzyme function. For example, sonicatesamplesatamedium-highsettingfor0,20,and30sec.ThenperformproteinorenzymefunctionassaysandmeasuretheA280ofeachsample.
b.Performthelysisorsonicationprocedureonice.
F. Reuse
1.Bindingdropsbeloworiginalcapacity • Lysate contains naturally occurring reducing agent or a nonspe-
cific polyanion may be obscuring the metal binding sites. a.Usealargervolumeofpreviouslyusedresin. b.Replaceusedresinwithfreshresin. c. Washresinwith6Mguanidinium(pH5.0)+1%TritonX-100
orSDS,andre-equilibratebeforeuse.
G. Application of samples prepared from overnight cultures to TALON Magnetic Beads.
1.Noproteinbindstothebeadswhenusingovernightculture.CheckthepHandensurethatitisbetween7–8.
2.Thebeadsfailtomigratetothemagnet,duetothehighviscosityofthesolution.
a. AddsufficientDNase(1unit/mlofculture)andmixthoroughlybeforeaddingbeads.
b. Dilutethesamplefurtherwith5XEquilibration/WashBuffertoobtainafinalconcentrationof1XEquilibration/WashBuffer.
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Bradford,M.M.(1976)Arapidandsensitivemethodforthequantificationofmicrogramquanti-tiesofproteinutilizingtheprincipleofprotein-dyebinding.Anal. Biochem.72:248–254.
Bush,G.L.,Tassin,A.-M.,Friden,H.&Meyer,D.I.(1991)Secretioninyeast:purificationandin vitrotranslocationofchemicalamountsofprepro-alpha-factor. J. Biol. Chem.266:13811–13814.
Chaga,G.,Bochkariov,D.E.,Jokhadze,G.G.,Hopp,J.&Nelson,P.(1999)Naturalpoly-histi-dineaffinitytagforpurificationofrecombinantproteinsoncobalt(II)-carboxymethylaspartatecrosslinkedagarose.J. Chromatogr. 864:247–256.
Hemdan,E.S.&Porath,J.(1985a)DevelopmentofImmobilizedMetalAffinityChromatographyII.Interactionofaminoacidswithimmobilizednickeliminodiacetate.J. Chromatogr. 323:255–264.
Hemdan,E.S.&Porath,J.(1985b)DevelopmentofImmobilizedMetalAffinityChromatographyIII.Interactionofoligopeptideswithimmobilizednickeliminodiacetate.J. Chromatogr.323:265–272.
Hochuli,E.,Döbeli,H.&Schacher,A.(1987)Newmetalchelateadsorbentselectiveforproteinsandpeptidescontainingneighboringhistidineresidues.J. Chromatogr.411:177–184.
Hochuli, E., Bannwarth,W., Döbeli, H., Gentz, R. & Stüber, D. (1988) Genetic approach tofacilitatepurificationofnovel recombinantproteinswithanovelmetalchelateadsorbent.Bio/Technology 6:1321–1325.
Kasher,M.S.,Wakulchik,M.,Cook,J.A.&Smith,M.(1993)One-steppurificationofrecom-binanthumanpapillomavirusType16E7oncoproteinanditsbindingtotheretinoblastomageneproduct.BioTechniques14:630–641.
Porath, J. (1985) Immobilized Metal IonAffinity Chromatography—A Powerful Method forProteinPurification.InModern Methods in Protein Chemistry,(pp.85–95).H.Tschelsche(Ed.),(Berlin&NY:WalterdeGruyter&Co).
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Sulkowski,E.(1985)PurificationofproteinsbyIMAC.Trends Biotechnol.3:1–7.
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XI. References
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 �4 Version No. PR6Z2142
TALON® Metal Affinity Resins User Manual
Appendix A. Reagent Compatibilities and Incompatibilities
A. Compatible reagents TableIIIshowsthemaximumconcentrationsofeachreagenttestedat
Clontech.Higherlevelsmaybeacceptable,buttheyshouldbetestedbeforeuse.Notethatsomeofthesereagentsmaypartiallyorcom-pletelydenatureyourprotein.
table iii. reagent compatibility
Reagent Acceptable Concentration
β-Mercaptoethanola 10mM(withcaution) CHAPSb 1%(withcaution) Ethanolc 30% Ethyleneglycol 30% HEPES 50mM Glycerol 20% Guanidiniuma 6M imidazoled 200mMatpH7.0–8.0,forelution KCl 500mM MES 20mM MOPS 50mM NaCl 1.0M NP-40 1% SDSb 1%withcaution TRISe 50mM Triton-X100 <1% Urea 8Ma Useresinimmediatelyafterequilibratingwithbufferscontainingthesereagents.Otherwise,
theresinwillchangecolor.Donotstoreresininbufferscontainingthesereagents.b IonicdetergentslikeCHAPS(3-[(30Cholamidopropyl)-dimethylammonio]-1-propane-sulfo-
nate),SDS(sodiumdodecylsulfate),andsarkosylarecompatibleupto1%.However,duetotheirchargednature,youshouldanticipateinterferencewithbinding.
c Ethanolmayprecipitateproteins,causinglowyieldsandcolumnclogging.d Imidazolecannotbeusedatconcentrationshigherthan5–10mMforloadingpolyhistidine-
taggedproteins,becauseitcompeteswiththehistidinesidechains(imidazolegroups)forbindingtotheimmobilizedmetalions.
e TRIScoordinatesweaklywithmetalions,causingadecreaseincapacity.
Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 ��
TALON® Metal Affinity Resins User Manual
B. Incompatible reagents Thesereagentsareincompatibleatanyconcentration:
• DTT(dithiothreitol)andDTE(dithioerythritol) Note:Useofstrongreducingagentswillinterferewiththebindingofthecobalt
metalionstotheresin.
• EDTA(ethylenediaminetetraaceticacid)andEGTA(ethyleneglycol-bis([β-amino-ethylether])
Note:AlthoughyoucanuseEDTAatindicatedpoints,itmustberemovedfromthesamplebygelfiltrationpriortoapplyingittoTALON®Resins.
Appendix A. Reagent Compatibilities and Incompatibilities
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 �6 Version No. PR6Z2142
TALON® Metal Affinity Resins User Manual
Appendix B. Mini-Scale Protein Purification Protocol
Mini-scaleproteinpurificationisidealforanyofthefollowing: (a)checkingforapolyhistidine-taggedprotein (b)determiningexpressionlevels (c)testingbufferconditionsYoucanuseaTALON®SingleStep(Cat.No.635628or635631)forproteinminiprep,oryoucanuseaTALONspin™Column(Cat.No.635601)withthisprocedure.Werecommendthatyousetasideasampleaftereachcriticalstepoftheprocedure,andanalyzeallsamplesbySDS/PAGE.Important• Thisprotocolisnotintendedforobtaininghighlypurifiedpolyhistidine-
taggedproteinsamples.Furthermore,proteinsampleselutedwithEDTA(Step19,below)willcontaincobaltandEDTA,whichmayseriouslyinhibitenzymeactivityandmaycausetheproteintoprecipitate.
• ThisprotocolwasoptimizedusingdenaturingconditionsatpH8.0.Ifyouwishtoobtainnativesamples,thensubstitutebuffersaccordingly.Youmayalsoneedtouselysozyme(0.75mg/mlofnativebuffer)tocompletelydisruptthecellsinStep5.
1.Transfer1mlofexpressionculturetoa1.5mlmicrocentrifugetube. 2.Centrifugeat14,000rpmfor2min. 3.Removeanddiscardsupernatant. 4.Add0.5mlofDenaturingEquilibrationBuffer(pH8.0). 5.Vortexuntilcellpelletiscompletelydissolved. 6.Centrifugeat14,000rpmfor5mintopelletanyinsolubledebris. 7.Setaside50µlofthesupernatantforlateranalysis.Transferthe
remainderofthesupernatanttoaclean1.5mltubecontaining50µlofprewashedTALON®Resin,preparedasdescribedinSectionVIII.B.Steps1–7.Startwith100µlofresuspendedslurry.
8.Agitatesampleatroomtemperaturefor10min. 9.Centrifugeat14,000rpmfor1mintopelletprotein/resincomplexes. 10.Carefully remove the supernatant and set aside 50 µl for later
analysis.Ahighproteinconcentrationinthissampleindicatesaproblemwithproteinbinding.
11.Add1mlofDenaturingEquilibrationBuffer. 12.Vortexforafewsec. 13.Centrifugeat14,000rpmfor1mintopelletresin. 14.Removethesupernatantandsetaside50µl(“firstwash”)forlater
analysis.Discardtheremainderofthesupernatant.
Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 ��
TALON® Metal Affinity Resins User Manual
Appendix B. Mini-Scale Protein Purification...continued
15.RepeatSteps11–14.Setaside50µlforanalysis. 16.Eluteboundpolyhistidine-taggedproteinbyadding50µlofElution
Buffertotheresin/proteinpelletandbrieflyvortexing. 17.Centrifugebrieflyat14,000rpm. 18.Carefully remove the supernatant containing the polyhistidine-
taggedprotein. 19.RepeattheSteps16–18.Alternatively,ifyouonlyintendtodetermine
theconcentrationofpolyhistidine-taggedproteininyoursample,youcanachieveamorecompleteelution,andthus,amoreac-curateproteinquantificationbyelutingwithEDTAasfollows:
a. Add50µlof100mMEDTA(pH8.0)andvortexbriefly. b. Centrifugebrieflyat14,000rpm. c. Carefullyremovethesupernatantcontainingthe6xhistidine-
taggedprotein. Note:EDTAremovesboundmetalfromtheresin:theproteinsamplewillcontain
cobalt,andtheTALON®Resincannotbereused.
20.Add12µlof5XSDS/PAGEsamplebuffertoeachofthesavedsamples. Note:Thesamplebufferwillreducemultimerstomonomers;thus,onlyasingleband
willbevisibleonanSDS/PAGEgel,evenfornaturallyhomologousmultimericproteins.
21.Heatsamplesat95–98°Cfor5min. 22.LoadsamplesandanalyzeonanSDS/PAGEgel.
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 �� Version No. PR6Z2142
TALON® Metal Affinity Resins User Manual
Figure 6. pHAT10/11/12 combined vector map and MCS. Uniquerestrictionsitesareinbold.ThesequenceofpHAT10isshown.TheasteriskindicatestheinsertionpointofadditionalbasesinpHAT11(G)andpHAT12(GG)thatalterthereadingframeoftheMCS.ThesevectorsencodeanovelpolyhistidineepitopetagthatenablespurificationofexpressedproteinsatneutralpH.ThepHATVectorsallowproteinpurificationunderbothnativeanddenaturingconditions.TheHATepitopeisanaturallyoccurring,19-amino-acidsequencefromthechickenlactatedehydrogenaseprotein.Thissequenceofnonadjacenthistidineresidueshas loweroverallchargethantagswithconsecutivehistidineresidues,suchasthe6xhistidinetag.Asaresult,HAT-proteinfusionsexhibitsolubilitythatmorecloselyresembleswild-typeproteinswhilestillpossessingstrongaffinityforimmobilizedmetalions.TheuniquebindingcharacteristicsoftheHATsequenceallowbothimidazole-andpH-gradientpurificationofproteinsundernativeconditionsatneutralpH(7.0),aswellasunderdenaturingconditions.TheHATsequenceandanenterokinase(EK)cleavagesitehavebeenincorporatedintothepUC19backbone.TheEKsiteallowsforoptionalremovaloftheHATsequencefromthepurifiedproteinbytreatmentwithenterokinase.RestrictionsitesallowexcisionoftheHATsequence,withorwithouttheEKsite,forcloninginothervectors.
Appendix C: Vector Information
pHAT10/11/122.� kb
MCS
Ampr
Plac
pUCori
HAT
EKsite
A AGC TTG AAG GAT CAT CTC ATC CAC AAT GTC CAC AAA GAG GAG CAC GCT CAT GCC CAC AAC AAG
ATCGATGACGATGACAAAGTCGACGGATCCCCGGGTACCGAGCTCGTAATTAGCTGAATTCSal I Sma I Sac IKpn IBamH I EcoR I
Hind III
Cla I
Ser Leu Lys Asp His Leu Ile His Asn Val His Lys Glu LysAsnHisHis Ala HisAlaGlu
EK cleavage site
HAT
*
Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 ��
TALON® Metal Affinity Resins User Manual
Appendix C: Vector Information continued
Figure 7. pHAT20 combined vector map and MCS. Uniquerestrictionsitesare inbold.ThesequenceofpHAT20isshown.ThisvectorencodesanovelHistidineAffinityTag(HAT)thatenablespurificationofexpressedproteinsatneutralpH.ThepHATVectorsallowproteinpuri-ficationunderbothnativeanddenaturingconditions.TheHATepitopeisanaturallyoccurring,19-amino-acidsequencefromthechickenlactatedehydrogenaseprotein.Thissequenceofnonadjacenthistidineresidueshasloweroverallchargethantagswithconsecutivehistidineresidues,suchasthe6xhistidinetag.Asaresult,HAT-proteinfusionsexhibitsolubilitythatmorecloselyresembleswild-typeproteinswhilestillpossessingstrongaffinityforimmobilizedmetalions.TheuniquebindingcharacteristicsoftheHATsequenceallowbothimidazole-andpH-gradientpurificationofproteinsundernativeconditionsatneutralpH(7.0),aswellasunderdenaturingconditions.TheHATsequenceandanenterokinase(EK)cleavagesitehavebeenincorporatedintothepUC19backbone.TheEKsiteallowsforoptionalremovaloftheHATsequencefromthepurifiedproteinbytreatmentwithenterokinase.RestrictionsitesallowexcisionoftheHATsequence,withorwithouttheEKsite,forcloninginothervectors.
pHAT202.� kb
Ampr
MCS (221–256)
pUC ori
Plac
ApaL I(481)
EK site
Hind III (141)
ApaL I(978)
ApaL I(2224)
EcoR I (262)
HAT
A AGC TTG AAG GAT CAT CTC ATC CAC AAT GTC CAC AAA GAG GAG CAC GCT CAT GCC CAC AAC AAG
ATC GAT GAC GAT GAC AAA
Hind III
Ser Leu Lys Asp His Leu Ile His Asn Val His Lys Glu LysAsnHisHis Ala HisAlaGlu
HAT
Cla I
EK cleavage siteGTT AAC CGG TCC CCG GGT ACC GGG CCC GGC CGG CCHpa I Age I Sma I Kpn I
Eag I Fse INae I
150•
160•
170•
180•
190•
200•
230•
240•
250•
204•