Living Colors User Manual Volume II - Molecular · PDF fileto the Indian and Pacificoceans...

Living Colors®

User Manual Volume II:Reef Coral Fluorescent ProteinsAmCyanAsRedDsRedDsRed-ExpressHcRedZsGreenZsYellow

PT3404-1 (PR37085)Published 17 July 2003

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 � Version No. PR37085

Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins

Table of Contents

I. Introduction 4

II. Properties of Reef Coral Fluorescent Proteins 6

A. StructureandFormationoftheRCFPFluorophore 6

B. PhotophysicalPropertiesofRCFPProteins 6

C. Discosomasp.RedFluorescentProtein(DsRed)andItsVariants 8

D. Heteractis crispa Far-RedFluorescentProtein(HcRed1) 12

III. Expression of Reef Coral Fluorescent Proteins 13

A. SuitableHostOrganismsandCells 13

B. VectorsforExpressingReefCoralFluorescentProteins 13

C. ExpressioninMammalianCells 15

D. SequencingPrimersforConfirmingIn-FrameFusions 15

IV. Detection of Reef Coral Fluorescent Proteins 16

A. Microscopy 16

B. FlowCytometry 19

C. DetectingFluorescentProteinsinEthanol-TreatedCells 20

D. AntibodiesfortheDetectionofRCFPs 20

V. Purified Recombinant Reef Coral Fluorescent Proteins 22

A. GeneralInformation 22

B. WesternBlotting:GeneralGuidelines 23

VI. Troubleshooting Guide 24

VII. References 25

VIII. Related Products 28

List of Figures

Figure1. ExcitationandemissionspectraofLivingColorsReefCoralFluorescentproteins 5

Figure2. GenealogyofDsRedproteins. 8

Figure3. TheFluorescentTimerProtein(DsRed1-E5)changesfromgreentoredovertime 10

Figure4. Recommendedcolorcombinations 18

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List of Tables

TableI. Generalinformationandnomenclature 5

TableII. PhotophysicalpropertiesofReefCoralFluorescentProteins 7

TableIII. Acomparisonofthepointmutationsin DsRed2&DsRed-Express 9

TableIV. LivingColors®ExpressionVectors 14

TableV. FiltersetsrecommendedforthedetectionofRCFPs 16

TableVI. DetectionofRCFPsbyflowcytometry 19

TableVII. Typicalinstrumentconfigurations 20

TableVIII.LivingColorsAntibodies 21

TableIX. Generalpropertiesofrecombinantredfluorescentproteins 22

TableX. SDS-PAGEandWesternblotdetection 23

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Table of Contents continued

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I. Introduction

Living Colors® Reef Coral Fluorescent Proteins (RCFPs)provideavaluable,non-invasiveapproachforinvestigatingbiologicaleventsinlivingcellsandtis-sues.Rangingincolorfromcyantofarred,RCFPscanbeusedtovisualize,track,andquantifymanydifferentcellularprocesses,includingproteinsynthesisandturnover,proteintranslocation,geneexpression,andcelllineage.Becausetheyrequirenoadditionalsubstratesorcofactorsfortheirfluorescence,ReefCoralFluorescentProteinsareidealforuseinlivecellassays.Andbecauseoftheirdistinctivespectra,theycanbereadilymultiplexed—thatis,combinedforthesimultaneousdetectionoftwoormoreeventsinthesamecellormixedcellpopulation.

Origins of Reef Coral Fluorescent ProteinsReefCoralFluorescentProteinsarederivedfromagroupofreefcoralsnativetotheIndianandPacificoceans(Matzet al., 1999).Thewild-typecDNAswereoriginallyisolatedbyMatzandcoworkers,whilesearchingforGFPhomologuesin the colored body parts of Anthozoan corals (Matz, et al., 1999). Using aPCR-based3'-RACE(RapidAmplificationof3'cDNAends)methodandasetofdegenerateprimerscorrespondingtodifferentdomainsinAequorea victoriaGreenFluorescentProtein(GFP),theyamplifiedanumberofcDNAsencodinga group of GFP-like fluorescent proteins, now known asAmCyan, ZsGreen,ZsYellow,DsRed,AsRed,andHcRed(Matz,et al.,1999;Lukyanov,et al., 2000;andGurskaya,et al., 2001).

Optimized for bright fluorescence and fast chromophore maturationToadapttheseproteinsforuseasin vivo reporters,aseriesofmutationshavebeenintroducedintothecorrespondingfull-lengthcDNAstoproduceRCFPswithhighersolubility,brighteremission,andmorerapidchromophorematuration.Inaddition,humancodon-optimizedversionsofeachcDNAhavebeencreatedforefficienttranslationinmammaliancells(TableI).

SpecificinformationconcerningthederivationandoptimizationofeachRCFPcan be found in the corresponding Vector Information Packet, provided witheachvectorandavailableonourwebsite.ThesepacketsdescribetheessentialfeaturesofthecorrespondingRCFP—includingthepositionandeffectofcertainkeymutations—andtheyciteliteraturereferenceswhenavailable.



0

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AmCyan1ZsGreen1

ZsYellow1 DsRed2AsRed2

HcRed1

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Wavelength (nm)

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AmCyan1 ZsGreen1ZsYellow1

DsRed2AsRed2

HcRed1

Excitation

Figure 1. Excitation and emission spectra of Living Colors® Reef Coral Fluorescent Proteins. Panel A.Excitation.Panel B.Emission.Curvesarenormalized;theheightofeachcurveisnotanindicationofrelativesignalstrengthperfluorophore.ThespectraforDsRed-Express(notshown)aresimilartothoseforDsRed2.

TABLE I. GENERAL INFORmATION AND NOmENCLATuRE

RCFPa Species of origin Human codon- Notable propertiesb

optimized

AmCyan Anemonia majano No BrighterthanECFP;AmCyan1 Yes photostablealternativeto ECFP

ZsGreen Zoanthus sp. No BrighterthanEGFPZsGreen1 Yes

ZsYellow Zoanthus sp. No Trueyellowemission;idealZsYellow1 Yes formultcolorapplications

DsRed-Express Discosoma sp. Yes PreferredDsRedforFACSc

DsRed2 Discosoma sp. Yes Increasedsolubilityvariant ofDsRed1

AsRed2 Anemonia sulcata Yes

HcRed1 Heterectis crispa Yes Far-redfluorescence; highlysoluble;dimer

a TheaminoacidsequencesofAmCyanandAmCyan1areidentical,onlythecodonusagehasbeenchangedtoenhancethetranslationinmammaliancells.Similarly,ZsGreenhasthesameaminoacidsequenceasZsGreen1;andZsYellowhasthesameaminoacidsequenceasZsYellow1.

b ECFPandEGFP=EnhancedCyanandGreenFluorescentProteins,respectively.c BecauseofthelowlevelofresidualgreenemissionfromDsRed-Express,cellsexpressingthis

proteinarereadilyseparatedfromthoseexpressingatruegreenfluorescentproteinsuchasEGFPorZsGreen.

I. Introduction continued

A B

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A. Structure and Formation of the RCFP FluorophoreTheRCFPfluorophoreisbelievedtoconsistofacyclizedtripeptidelocatedonthecentralα-helixinthecoreoftheprotein.Itformsautocatalytically,withinseveralhoursofproteinsyntheisbywayofanintramolecularreactioninvolvingahighlyconservedsetofaminoacidsidechains,justasinGFP(Matzet al., 1999;Grosset al.,2000).Theformationofthefluorophoredoesnotrequireanyotheragentsexceptmolecularoxygen.

B. Photophysical Properties of RCFP ProteinsRCFPsrangeincolorfromcyantofar-red(TableII).Ingeneral,theyhavebroadexcitationspectraandnarrowemissionspectra(Figure1).LiketheenhancedcolorvariantsofAequorea GFP,RCFPscanbedetectedincellsandtissueswithouthavingtoaddadditionalcofactorsorsubstrates,andtheyare extremely stable,making it easy tomonitor fluorescenceoverextendedperiodsoftime.RCFPmonomerssharestructuralhomology toAequorea victoria greenfluorescentprotein(Wallet al.,2000;Yarbroughet al.,2001).UnlikeGFP,however,RCFPmonomersselfassociateinsolutiontoformhigherordermultimerssuchasdimersandtetramers.Mostarebelievedtobeobligatetetramerswithstructuressimilartothatofwild-typeDsRed(Yarbroughet al.,2001).TheonlyexceptionsofarnotedisHcRed1,showntobeadimer(Gurskayaet al., 2001).Becauseof theirmultimericstructuresand their tendency toaggregate,RCFPsarenotgenerallyrecommendedforuseasfusiontags.Still,manypublishedstudiesshowthatRCFPscanbefusedtoaproteinofinterestwithoutinterferingwithitsnormalbiologicalfunction(Shulzet al., 2002;Nelsonet al., 2002;Engqvist-Goldsteinet al., 2001;Tsuboiet al., 2002;Nechushtanet al., 2001).SomeRCFPssuchasDsRed2andDsRed-Express,improvedthroughmutagenesis,areconsideredtobemoresoluble,andthereforelesspronetoaggregation, thanotherRCFPs(TableII).Nevertheless, ifyouareprimarilyinterestedinconstructingfluorescentfusionproteinsforlocalizationstudies,wesuggestyoufirsttryoneofourenhancedGFPcolorvariants:ECFP,EGFP,orEYFP.Ifdesired,compareyourresultstothoseobtainedwithacorrespondingRCFPfusiontofindoutwhethertheRCFPinterfereswiththefunctionordistributionoftheprotein.

I. Properties of Reef Coral Fluorescent Proteins



II. Properties of Reef Coral Fluorescent Proteins continued

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C. Discosoma sp. Red Fluorescent Protein (DsRed) and Its VariantsWildtypeDiscosoma sp. redfluorescentprotein,knowncommerciallyasDsRed,wasoriginallyisolatedbyMatzet al. (1999),whoreferredtotheproteinasdrFP583.Sincethen,numerousvariantshavebeendeveloped,including DsRed2, DsRed-Express, and DsRed1-E5—the FluorescentTimer(Figure2).Wild-typeDsRedhasbeenextensivelystudiedandcharacterized(Bairdet al.,2000;Grosset al.,2000;Heikalet al.,2000;Jakobset al.,2000;Vrzheshchet al., 2000;Wallet al.,2000).MuchoftheworkhasbeendevotedtolearningmoreabouttheDsRedfluorophore:itsstructure,aswellasitslightabsorbingandemittingproperties.

DsRed (wild-type)

DsRed1(human codon-optimized)

DsRed1-E5(Fluorescent Timer)

DsRed-Express(rapid maturation;

reduced aggregation;reduced green emission)

DsRed-Express-DR(destabilized reporter)

DsRed2(accelerated maturation;

reduced aggregation)

Figure 2. Genealogy of DsRed proteins.


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1. DsRed2DsRed2isafaster-maturing,moresolublevariantofDsRed1(July2001Clontechniques).Itcontainssixaminoacidsubstitutions(TableIII),whichresult inthemorerapidappearanceofredfluorescenceandreducetheprotein’s tendency to aggregate. Although it probably forms the sametetramericstructureasDsRed1(Yarbroughet al., 2001),DsRed2islessprone to forming insoluble aggregates, often observed in bacterial andmammaliancellsystemsexpressingDsRed1.

2. DsRed-ExpressDsRed-ExpressisavariantofDsRed1improvedthroughacombinationofsite-directedandrandommutagenesis(Bevis,B.J.,et al., 2002).Itcontainsnineaminoacidsubstitutions(TableIII),whichnotonlyimprovetheprotein’smaturationandsolubility,butalsoreducethelevelofresidualgreenemission,makingDsRed-ExpressthepreferredDsRedforflowcytometry.Thereductioningreenfluorescenceallowsforcompleteseparationofred-emittingandtruegreen-emittingpopulations(July2002Clontechniques). ThoughDsRed-ExpressappearstohavealowerquantumyieldandextinctioncoefficientthaneitherDsRed2orDsRed1(Bevis,B.J.,et al., 2002),itslowerintensityisoffsetbyitsacceleratedmaturation,whichultimatelyresultsinamorerapidbuild-upofredfluorescenceinthecell.

TABLE III. A COmPARISON OF THE POINT muTATIONS IN DsRed2 & DsRed-Express

DsRed2 DsRed-Express Effect

R2A R2A K5E K5E Enhancedsolubility K9T N6D

V105A T21S I161T H41T Acceleratedmaturation S197A N42Q V44A

C117S Reducedgreenemission T217A




3. DsRed1-E5 (Fluorescent Timer)DsRed1-E5,theFluorescentTimer,avariantofDsRed1,containstwoaminoacid substitutions (V105A and S197T) which increase its fluorescenceintensityandendowitwithadistinctspectralproperty:Astheproteinages,itchangescolor(Figure3;Terskikhet al.,2000).Shortlyafteritssynthesis,DsRed1-E5beginsemittinggreenfluorescence.Butastimepasses,thefluorophoreundergoesadditionalchangesthatshiftitsfluorescencetolongerwavelengths—whenfullymaturedtheproteinisbrightred.Inmammaliancells transfectedwithaTet-inducibleDsRed1-E5expressionvector, thegreen-to-redtransitionstartsabout3hoursaftertheproteinfirstbecomesfluorescent(Terskikhet al.,2000).Theprotein’spredictablecolorshiftcanbeusedtofollowtheonandoffphasesofgeneexpression(e.g.,duringembryogenesisandcelldifferentiation).AndwithDsRed1-E5,thegreenandredemissionsareeasilydetectedbyfluorescencemicroscopyandflowcytometry.DuringthefirstfewhoursofDsRed1-E5expressionin vivo,hostcellsappeargreen.Continuedexpressionresults in a mixture of green and red fluorescence—host cells appearyellowwhentheemissionsareoverlayed.Ifexpressionstops,hostcellswillgraduallyturnred.ThusDsRed1-E5letsyoutracknotonlyup-regulation,butalsodown-regulationofgeneexpression.FormoreinformationaboutthepropertiesandutilityofDsRed1-E5,pleaseseeTerskikhet al.(2000)andApril2001Clontechniques.

500 nm

Time (hr)

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ence

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Figure 3. The Fluorescent Timer (DsRed1-E5) changes from green to red over time. Measure-mentsweremadeusingrecombinantDsRed1-E5,freshlypurifiedfromanovernightE. coli culture.Theproteinwaspurifiedat4°CusingaTALONTMMetalAffinityResinandice-coldbuffers.Purifiedproteinwasstoredoniceuntilt =0hr,whenitwasplacedat37°Candthefirstmeasurementwasrecorded.




4. DsRed-Express-DR (destabilized reporter)DsRed-Express-DRisadestabilizedvariantofDsRed-Express.Incontrasttotheoriginalprotein,whichisextremelystable,DsRed-Express-DRhasashorthalf-life,makingitwellsuitedforstudiesthatrequirerapidreporterturnover.DsRed-Express-DRwasconstructedbyfusingtheC-terminusoftheproteintoaminoacidresidues422–461ofmouseornithinedecarboxylase(MODC),oneofthemostshort-livedproteinsinmammaliancells(Li,X.,et al., 1998).ThisregionofMODCcontainsaPESTsequencethattargetstheproteinfordegradation,resultinginrapidproteinturnover(Li,X.,et al., 1998;Rechsteiner,M.,et al., 1990).DsRed-Express-DRcanbeusedasanin vivoreporterofgeneexpression(Clontechniques October 2002). Because of its rapid turnover rate, itsexpressionfromapromoterofinterestprovidesamoreaccurateassessmentof the promoter’s activity over time than does the more stable DsRed-Express. Other destabilized RCFP vectors include pZsGreen1-DR andpHcRed1-DR.

5. How pH Affects DsRed FluorescenceDsRed’sabsorptionandemissionpropertieschangevery littlewithpH.According to Baird and coworkers, only extremely alkaline (≥pH12) ormoderatelyacidic(≤pH5)conditionsdepressDsRed’sfluorescence(Bairdet al., 2000).Formostin vitro studiesofDsRedwegenerallyuseaqueoussolutions(e.g.,10mMTris-HCl)bufferedbetweenpH8.0–8.5.

6. How Chemical Reagents Affect DsRed FluorescenceUnlikeAequorea victoria greenfluorescentprotein,DsRedfluorescenceissensitivetomilddenaturants,suchassodiumdodecylsulfate(SDS).FullydenaturedDsReddoesnotfluoresce.

7. DsRed Resists PhotobleachingDsRedresistsphotobleaching—itsfluorescenceremainsstableduringthetimestypicallyrequiredtomakemeasurementswithaspectrofluorometerorstandardfluorescencemicroscope(ClontechLaboratories,Inc.,unpublishedobservations;Bairdet al.,2000).


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D. Heteractis crispa Far-Red Fluorescent Protein (HcRed1) 1. HcRed1 HcRed1wasgeneratedbyrandomandsite-directedmutagenesisofa

non-fluorescenttetramericchromoproteinfromthereefcoralHeteractis crispa(Gurskayaet al., 2001).Earlyroundsofrandommutagenesiswereusedtoproducevariantswithextremefar-redfluorescenceandrapidmaturationrates.Afterisolatingthebrightestvariant,investigatorsusedsite-directedmutageneistooptimizetheprotein’ssolubility.Thefinalhumanizedvariant,HcRed1(HcRed-2AinGurskayaet al., 2001),wasselectednotjustbecauseofitsbrightfar-redfluorescence,butalsobecauseofitsapparentdimericnature.

Because of its far red-shifted fluorescence, HcRed1 is easilydistinguishedfromourotherfluorescentproteins,includingDsRed2,andDsRed-Express(April2002Clontechniques).Inoneexample,aFACSVantage™flowcytometerequippedwithanargon/kryptonlaser(whichemitsa568nmline)wasusedtoseparateamixedpopulationofcellsexpressingeitherDsRed2orHcRed1intospectrallydistinctpopulations (April 2002 Clontechniques). In another experiment,threesubcellular compartmentswere labeledandclearly visualizedby cotransfecting cells with localization vectors encoding HcRed1,ECFP,andEYFP(April2002Clontechniques).OfalltheRCFPsintheLivingColorsfamily,HcRed1istheonlymemberknowntobeadimer,andisthereforeconsideredtobethemostsuitablechoiceforproteinlocalizationstudies.

2. HcRed1-DR (destabilized reporter) HcRed1-DR is a destabilized variant of HcRed1. Its construction is

identicaltothatusedforDsRed-Express-DR(discussedabove).LikeDsRed-Express-DR, ithasashorthalf-life,making itwellsuited formeasuringchangesingeneexpression.




A. Suitable Host Organisms and CellsRCFPscanbeexpressedinawidevarietyofprokaryoticandeukaryoticcelltypes.Theyarewelltoleratedbymammaliancells,andhavethereforeproventobeusefulforcreatingstablytransfectedcelllinesandtransgenicorganisms(Handler&Harrell,2001;Fenget al., 2000).MammaliancelllinestestedsofarbyusincludeHEK293,HeLa,3T3,Jurkat,andHT1080.Others have reported that RCFPs can be expressed and detected inSaccharomyces cerevisiae, E. coli, C. elegans, Drosophila, Xenopus, Zebrafish(pers.communications),andmouse(Fenget al., 2000).Todate,DsRedand itsvariantsare themostwidelyusedRCFPs,andmanydifferentexamplesoftheirusecannowbefoundintheliterature.Forspecificexamples,wesuggestyousearchourcitationsdatabase,availableatwww.clontech.com,aswellasotherpublicdatabases forpublishedstudiesrelevanttoyourareaofinterest.

B. Vectors for Expressing Reef Coral Fluorescent ProteinsClontech offers a complete line of vectors for expressing RCFPs inmammaliancellsandbacteria(TableIV).Formoredetailedinformation,includingsequences,cloningsites,andplasmidmaps,pleaserefertothecorrespondingVector InformationPacketprovidedwitheachvectorandavailableonourourwebsiteatwww.clontech.com.

1. Bacterial expression vectors OurbacterialvectorsareprimarilyintendedtoserveassourcesofRCFP

cDNA.MCSregionsflankthecDNAinsertsothatitcanbeeasilyexcisedandshuttledintootherexpressionsystems.ThesevectorscanalsobeusedinbacteriaforbulkproductionoftheRCFPprotein.Expressionisdrivenbythelacpromoter(Plac)and,therefore,canbeinducedbyisopropylthio-β-d-galactoside(IPTG).Theproteinsareexpressedasfusionswithseveralaminoacids,includingthefirstfiveaminoacidsoftheLacZ protein.EachvectorcontainsanampicillinresistancegeneandthepUCori.

2. mammalian expression vectors Our fusion vectors contain the immediate-early promoter from

cytomegalovirus(CMVIE)forstrongconstitutiveexpressionofthegenein vivo.TheyalsocontainanupstreamKozakconsensussequencetofurtherenhancethetranslationefficiency.ThesevectorscanalsobeusedastransfectionmarkerssincetheemptyvectorwillexpressRCFP.

III. Expression of Reef Coral Fluorescent Proteins



TABLE IV. LIVING COLORS® ExPRESSION VECTORS

Vector Type Host Promoter Selection uses/Purpose(naming convention) Prok. Euk.

All RCFPS

Fusion Mammalian CMVIE Kan Neo Expressaproteinofinterestas(pRCFP-N1,or afusiontotheN-orC-terminuspRCFP-C1) ofanyRCFP

Promoterless Mammalian None Kan Neo Monitortranscriptionfrom(pRCFP-1) differentpromotersand promoter/enhancercombinations

Bacterial Bacterial lac Amp None Intendedprimarilyasaconvenient(pRCFP) sourceofthefluorescentprotein cDNA;flankingrestrictionsites allowforeasyexcisionofthe full-lengthcodingsequence.

Selected RCFPs

Subcellular Mammalian CMVIE Kan Neo Ready-to-usevectorsforlabelingLocalization varioussubcellularstructures:(pRCFP-structure) mitochondria,nucleus, endoplasmicreticulum,or peroxisomes.

Bicistronic Mammalian CMVIE Kan Neo Containaninternalribosomeentry(pIRES2-RCFP) site(IRES),whichpermits translationoftwoORFsfroma singlemRNA.Thuscells expressingthefluorescent proteinalsoexpressthegene ofinterestandcanbeselected byflowcytometry

III. Expression of Reef Coral Fluorescent Proteins cont.



C. Expression in mammalian Cells

Vectorsmaybetransfectedintomammaliancellsbyavarietyoftechniques,including those using calcium phosphate (Chen & Okayama, 1988),DEAE-dextran (Rosenthal, 1987), various liposome-based transfectionreagents (Sambrook et al., 1989), and electroporation (Ausubel et al.,1994). Any calcium phosphate transfection procedure may be used,but we recommend using the CalPhosTM Mammalian Transfection Kit(Cat.No.631312).Likewise,anyliposome-mediatedtransfectionproceduremay be used. For further information on cell culture techniques, seeFreshney(1993).

The efficiency of a mammalian transfection procedure is primarilydependenton thecell line.Therefore,whenworkingwithacell line forthe first time, we recommend you compare the efficiencies of severaltransfection protocols. This test can best be accomplished usingpDsRed-Express-CMV,whichhastheCMVimmediateearlypromoterforhigh-levelexpressioninmostmammaliancelllines.Thelengthoftimerequiredforfluorescencetoinitiallybecomedetectableandthetotalelapsedtimeneededtoattainthemaximumfluorescentsignalinagivencellpopulationfollowingtransfectionwilldepend,inpart,ontheparticularcelllineused.Inmostcases,DsRed-Expressmaybedetectedbyfluorescencemicroscopy,FACSanalysis,orfluorometrywithin8–12hoursoftransfection.

Asanalternativetostandardtransfection,youmaywanttotryoneofourviral-mediatedgenetransferandexpressionsystems.Weofferadenoviral,retroviral,andbaculoviralbasedsystemsforexpressingproteinsinmanydifferent cell types.Adenovirus, for example, enables you to efficientlyinfect bothdividingandnon-dividingcell lines.Youcanconstruct yourownrecombinantadenovirususingaAdeno-XExpressionSystem,oruseoneofourpremadeAdeno-XMarkerViruses,expressingeitherEGFPorDsRed2(seeRelatedProducts).

D. Sequencing Primers for Confirming In-Frame FusionsTosequencejunctionsupstreamordownstreamoftheDsRed,DsRed1,DsRed2,andDsRed-Expresscodingregions:

• DsRed-NSequencingPrimer(Cat.No.632386) Forwild-typeDsRedN-terminalgenefusions • DsRed-CSequencingPrimer(Cat.No.632389) Forwild-typeDsRedC-terminalgenefusions • DsRed1-NSequencingPrimer(Cat.No.632387) ForDsRed1,DsRed2,andDsRed-ExpressN-terminalgenefusions • DsRed1-CSequencingPrimer(Cat.No.632388) ForDsRed1,DsRed2,andDsRed-ExpressC-terminalgenefusions

III. Expression of Reef Coral Fluorescent Proteins cont.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 1� Version No. PR37085


A. microscopy 1. Conventional Fluorescence microscopy a. Filter sets TableV,below,listsfiltersetssuitableforthedetectionofRCFPs

byfluorescencemicroscopy.Althoughyoucanachievesatisfactoryresults using standard filters sets, such as FITC filters to detectZsGreenandrhodamineorpropidiumiodidefilterstodetectDsRedanditsvariants,optimizedfiltersetsforthedetectionofReefCoralFluorescentProteinshavebeendevelopedbyChromaTechnologyCorp(www.chroma.com)incollaborationwithClontechLaboratories,Inc..

TABLE V. FILTER SETS RECOmmENDED FOR THE DETECTION OF RCFPS

Optimized Filters from Chroma Technology Corp Standard Filters

RCFP Filter set name Excitation Dichroic Emission Common Name

AmCyan1 AmCyan D440/40x 470dcxr D500/40m ECFP

ZsGreen1 ZsGreen HQ470/35 Q490lp HQ520/40 FITC/EGFP

ZsYellow1 ZsYellow HQ500/40 530dclp HQ550/40m EYFP

DsRed2a DsRed D540/40x 570dclp D600/50m TRITC/Phycoerythrin

DsRed-Express DsRed D540/40x 570dclp D600/50m TRITC/Phycoerythrin

AsRed2a AsRed D540/40x 570dclp HQ620/60m TRITC/Phycoerythrin

HcRed1 HcRed HQ575/50x 610dclp HQ640/50m TexasReda TheDsRed2andAsRed2filtersetsareinterchangeable.

b. Light source Theemissionintensityfromalightsourcecanvarywithwavelength.

Because the intensity of the excitation light directly affects thebrightnessofa reporter, it is important tocompare theexcitationspectraofyourchosenreporterswiththeemissionspectrumofthelightsource.Conventionalfluorescencemicroscopesareequippedwitheitheramercury-orxenon-arclamp.Bothlampsaresuitableforexcitingfluorescentproteins.Butbeaware that,whereas thelightintensityfromaxenonlampdeviatesverylittleacrosstheblue,green,yellow,andredregions,theemissionfromamercurylamphassharppeaksinthecyanandredregions.Therefore,fluorophoresexcitedbycyanandredlightmayappearbrighterwhenexcitedbyamercurylamp.Checkthemanufacturers’specificationsfordetailsaboutyourlamp(s).

IV. Detection of Reef Coral Fluorescent Proteins



c. Detection systems Inconventionalmicroscopy,fluorescenceisusuallydetectedwith

eitheraphotographic(35-mm)ordigitalcamera(whichfrequentlyharborsacooledcharge-coupleddevice,orCCD).Recordinganimage with a 35-mmcameramay require a longer-than-averageexposuretime,possiblyphotobleachingthesample.Digitalcameras,ontheotherhand,areusuallymoresensitivethan35-mmcamerasbut(unlessyouuseacolordigitalcamera)requireimageanalysissoftwaretoproduce“pseudo-colored”data.

d. multicolor analysis Withthedevelopmentofoptimizedfiltersetsandtheintroduction

of our red fluorescent proteins, it’s now possible to separate asmanyasthreefluorescentreporters—cyan,yellow,andred—usingconventional fluorescence microscopy. Some investigators haveevendistinguishedallfourcolors(cyan,green,yellow,andred)usingflowcytometry(Hawleyet al., 2001;July2003Clontechniques).Infact,manyexamplesofmulticoloranalysiscannowbefoundintheliterature,andwerecommendyousurveytheliteratureforhelpindecidingwhichcombinationwillworkbestforyou.Here,weofferthe following recommendations for two-and three-coloranalyses(Figure4).

2. Laser-scanning microscopy See Table VI for laser lines recommended for the excitation of

RCFPs. 3. multi-photon laser-scanning microscopy Multi-photon excitation microscopy has won the approval of many

researcherswhoseekalternativesforresolvingfluorescentlylabeledproteinsin vivo.Itsadvantageshavebeenwidelyreported(Piston,D.W.,1999;Potter,S.M.,1996;Marchant,J.S.,2001).Chiefamongthemistheabilitytoexcitefluorophoreswithlow-energyinfraredlight.IR lightnotonlypenetrates tissuemoreeffectively thanvisible lightbutalsominimizesphotobleachingandcauseslessphotodamage.Inprinciple,allLivingColorsFluorescentProteinsaresuitableforuseinmulti-photonapplications.

IV. Detection of Reef Coral Fluorescent Proteins continued




ZsYellow1 HcRed1

DsRed2

DsRed-Express

AsRed2

HcRed1 ZsYellow1

AmCyan1 HcRed1

HcRed1 AmCyan1

AmCyan1

AmCyan1 ZsYellow1

ZsYellow1 AmCyan1

ZsGreen1

HcRed1

DsRed2

DsRed-Express

AsRed2

HcRed1

First Color Second Color Third Color

ZsGreen1

ZsYellow1

AmCyan1

ZsGreen1DsRed2

AmCyan1

ZsGreen1DsRed-Express

AmCyan1

ZsGreen1AsRed2

Figure 4. Recommended color combinations.

Protocol No. PT3404-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR37085 1�


B. Flow CytometryLaser lines and filter sets suitable for the detection of RCFPs by flowcytometryarelistedinTableVI.SeealsoTableVIIfortypicalinstrumentconfigurations.

TABLE VI. DETECTION OF RCFPS By FLOw CyTOmETRy

Excitation requirement EmissionRCFP Laser line (nm) Laser Filter /bandpass (nm)

AmCyan1 405 VioFlameSolidState 485/22or530/30 407 PointSourceSolidState 485/22or530/30 413 Krypton 485/22or530/30 458 Argon 485/22or530/30 458 Spectrum 485/22or530/30 488 Argon 530/30

ZsGreen1 488 Argon 519/20,520/40,or530/30

ZsYellow1 488 Argon 530/30,550/30,or585/42 514 Argon 550/30,or585/42 531 Spectrum 585/42

DsRed2 488 Argon 555/20,580/30,585/42,or610/20

514 Argon 555/20,580/30,585/42,or610/20

531 Spectrum 555/20,580/30,585/42,or610/20

568 Spectrum 610/20or630/30

DsRed-Express 488 Argon 555/20,580/30,585/42,or610/20 514 Argon 555/20,580/30,585/42,or610/20 531 Spectrum 555/20,580/30,585/42,or610/20 568 Spectrum 610/20or630/30

AsRed2 488 Argon 580/30,585/42,610/20,or630/30 514 Argon 580/30,585/42,610/20,or630/30 531 Spectrum 580/30,585/42,610/20,or630/30 568 Spectrum 610/20or630/30

HcRed1* 568 Spectrum 610/20,630/30,or660/20 633 HeNe 660/20 635 RedDiode 660/20*NotethatHcRed1cannotbeefficientlyexcitedbya488-nmlaserline.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 �0 Version No. PR37085


TABLE VII. TyPICAL INSTRumENT CONFIGuRATIONS

Instrument Laser Laser line (nm)

FACScan™System Argon(L1) 488

FACSCalibur™System Argon(L1) 488 RedDiode(L2) 635

FACStar™PlusSystem Argon(L1) 488(typicalsetup) HeNe(L2) 633

FACSVantage™SESystem Argon(L1) 488(typicalsetup) Krypton(L2) 407 HeNe(L2orL3) 633

LSRSystem Argon(L1) 488 (typicalsetup) HeCd(L2) 325 HeNe(L3) 633

LSRIISystem Argon(L1) 488(typicalsetup) HeNe(L2) 633 UV(L3) 355 Violet(L4) 405

FACSAria™Cell-Sorting Argon(L1) 488 System(typicalsetup) HeNe(L2) 633 Violet(L3) 407

FACSArray™Bioanalyzer GreenDiode(L1) 532 System RedDiode(L2) 635

C. Detecting Fluorescent Proteins in Ethanol-Treated CellsWhenexpressedassolubleproducts,fluorescentproteinsmayleakfromcellstreatedwithethanolormethanol.Topreventsuchlosswesuggestusingparaformaldehydeinsteadofethanolormethanol.

D. Antibodies for the Detection of RCFPsClontech offers a wide variety of monoclonal and polyclonalantibodies for the detection of Living Colors Fluorescent Proteins byWestern blotting, immunoprecipitation, and immunocytochemistry(TableVIII).TheantibodiesareespeciallyusefulforanalyzingN-andC-terminal fusions following theirexpression in vivo.BecauseRCFPsandAequorea victoria GFPvariantsarederivedfromdifferentorganisms,theantibodiesraisedagainstthesetwofamiliesdonotcrossreact.BecauseeachRCFPisauniqueproteinencodedbyadistinctgeneratherthanamutantvariantofasinglefluorescentprotein(asintheA. victoria family),it has been possible to develop highly specific antibodies for individualRCFPs(TableVIII).


Protocol No. PT3404-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR37085 �1



TAB

LE

VIII

. LIV

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LO

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® A

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Affi

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632

380

(IgG

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nalA

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Rab

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+

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men

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632

382

usin

gfu

ll-le

ngth

GF

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in

A.v

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Ant

ibod

y(p

olyc

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6

3237

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finity

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Ab'

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DsR

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nize

s

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3239

3D

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and

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632

397

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sva

riant

s

HcR

edP

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6324

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poo

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uite

d

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 �� Version No. PR37085


A. General InformationRecombinantReefCoralFluorescentProteins fromClontech (Table IX)are produced in E. coli, and then affinity purified by metal ion affinitychromatography(IMAC)usingTALON™Resin.Thepurifiedrecombinantproteinscontaina6xHN,N-terminalepitopetag.Thetagdoesnotinterferewiththeproteins’characteristicspectralproperties,butifyouwishtoremoveit,youcandosobydigestionwithenterokinase(EK);thereisasingleEKsitebetweenthetagandtheprotein.For an EK digestion protocol, please refer to the EK manufacturer’s guidelines.

Recombinant Reef Coral Fluorescent Proteins can be used as positivecontrols and as standards in studies involving their expression in vivo.Theirspectralpropertiesareidenticaltothoseoftheproteinsexpressedinmammaliancells.Youcanusethemasstandardstocalibratefluorometers,andaspositivecontrolstoverifyanalysisbySDS-PAGEandWesternblot,usingtheappropriateantibodies.Additionalapplicationsincludeisoelectricfocusing and fluorescence activated cell sorting (FACS). Longtermexpression studies of stably transfected mammalian cell cultures haveshowntheseproteinstobewelltolerated.Thus,itisreasonabletoexpectthattherecombinantproteins,thoughproducedinE. coli,aresuitableformicroinjectionintomammaliancellsandtissues.Please note, however, that we have not established specific protocols for using recombinant red fluorescent proteins in isoelectric focusing, FACS, or microinjection. InPartB,below,weoffer somegeneralguidelines forSDS-PAGEandWesternblotting.

TABLE Ix. GENERAL PROPERTIES OF RECOmBINANT RED FLuORESCENT PROTEINS

Protein mass (kDa)a Ex. max (nm) Em. max (nm) western blot analysisb

rDsRed-Express ~25.7 557 579 DsRedMonoclonalAntibody (Cat.No.632393)or DsRedPolyclonalAntibody (Cat.No.632397)

rDsRed2 ~25.7 563 582 DsRedMonoclonalAntibody DsRedPolyclonalAntibody

rHcRed1 ~25.7 588 618 HcRedPolyclonalAntibody (Cat.No.632452)

a Calculatedmassofmonomer.ThoughDsRed-Express,DsRed2,andHcRed1eachhaveacalculatedmassof~25.7kDa, theyusually runabove29kDaonSDS-polyacrylamidegels.DsRed2andDsRed-Expressarebelievedtoformthesametetramericstructureaswild-typeDsRed.HcRed1ismostlikelyadimer(Gurskaya,N.G.,et al., 2001).

b RecommendedLivingColorsantibody.

V. Purified Recombinant Reef Coral Fluorescent Proteins



B. western Blotting: General GuidelinesUseastandardprocedureforpolyacrylamidegelelectrophoresis(PAGE)to resolve theproteinsonaone-dimensionalgel (Laemmli,1970).Justpriortouse,allowtherecombinantproteintothawatroomtemperature,andthenplacethetubeonice.Prepareanaliquotoftheproteinaccordingto theprotocolappropriate foryourelectrophoresissystem.Gel loadingrecommendationsdependonthesizeofthegelandthemethodofdetection:CoomassiebluestainingorWesternblotting.SeeTableX,below.

TABLE x. SDS-PAGE AND wESTERN BLOT DETECTION

Loading Recommendations (amount of protein per lane)a

Apparatus method of detectionb Purified recombinant protein Cell/tissue lysate

Minigel Coomassiebluestaining ~0.5–1µg ~25–75µg

Minigel Westernblot ~5–50ng 200µg (antibody-baseddetection)

a Ifyouareaddingrecombinantfluorescentproteintoatotalcell/tissuelysateorothercrudesample,theamountoftotalproteinloadedperlanemustbeoptimizedfortheparticularapplication.

b Generally,LivingColorsrecombinantredfluorescentproteinswillnotfluoresceonanSDSgelorWesternblot.

V. Purified Recombinant Proteins continued



A. Potential Difficulties Encountered using RCFPs • Longdelaybetweentransfectionanddetectionoffluorescence Ifyoufailtodetectfluorescencefromconstructsinyoursystem,there

areseveralpossibleexplanations.Theseincludeuseofaninappropriatefilterset,expressionoftheRCFPbelowthelimitofdetection,andfailureoftheproteintoformthefluorophore.CheckyourvectorconstructtobesureyourgenewasinsertedinframewiththeRCFPgene.Someproteins,whenfusedtotheN-orC-terminusofanRCFP,couldinterferewith the formation of the chromophore or with the assembly of thefluorescentproteinintoitsoligomericstructure.TheexpressionofanRCFP fusion in vivo canbeconfirmedbyWesternblottingwith theappropriateantibody.

B. Considerations for mammalian Expression • Usevectorsexpressingthehumancodon-optimizedvariantoftheRCFP

youwishtoexpress. •Verifytheidentityofyourplasmidconstructandchecktheconcentration

witharestrictiondigest.Confirmthatallsubcloningstepshavebeendonecorrectly.Beawarethatspecificrestrictionsitesinsomevectorsareinactivatedbymethylation.

•CheckthetransfectionefficiencyofyourexperimentalsystemusingasecondreporterplasmidthatcontainsthesamepromoterelementsuchaspEGFP-N1 (Cat.No.632318).

• WhenusingRCFPstostudyproteinlocalization,beawareofpossibleperturbingeffects.Becauseoftheirproposedmultimericstructuresandtendencytoaggregate,RCFPsmayinterferewiththenormalfunctionand localization of your protein of interest.Therefore, we generallyrecommendyouuseoneofourenhancedGFPcolorvariantstomonitorproteinlocalization in vivo. Ifdesired,compareyourresultstothoseobtainedwithacorrespondingRCFPfusion.

C. Optimizing microscope/FACS Applications • Ingeneral,youcanseethefluorescencebetterinadarkenedroom

afteryoureyeshaveadjusted. •FiltersetsfordetectingRCFPsareavailablefromChromaTechnology

Corp(www.chroma.com). • DsRedfluorescencemaybesensitivetosomenailpolishesusedto

seal coverslips (though black nail polish does not appear to inhibitDsRedfluorescence).Insteadofnailpolishformountingcoverslips,werecommendmoltenagarose,rubbercement,oracommercialmountingsolutionsuchasProLong®AntifadeKit(MolecularProbes).

• Intense excitation of fluorescent proteins for extended periods maygeneratefreeradicalsthataretoxictolivingcells.Thisproblemcanbeminimizedbyusinglongerwavelengthstoexcitethefluorophore.

VI. Troubleshooting Guide



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VII. References

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Wall,M.A.,Socolich,M.&Ranganathan,R.(2000)ThestructuralbasisforredfluorescenceinthetetramericGFPhomologDsRed.Nat. Struct. Biol.7:1133–1138.

Yang,T.T.,Sinai,P.,Green,G.,Kitts,P.A.,Chen,Y.T.,Lybarger,L.,Chervenak,R.,Patterson,G.H.,Piston,D.W.&Kain,S.R.(1998)Improvedfluorescenceanddualcolordetectionwithenhancedblueandgreenvariantsofthegreenfluorescentprotein.J. Biol. Chem.273:8212–8216.

Yarbrough,D.,Wachter,R.M.,Kallio,K.,Matz,M.V.&Remington,S.J.(2001)RefinedcrystalstructureofDsRed,aredfluorescentproteinfromcoral,at2.0-Åresolution.Proc. Natl. Acad. Sci. USA 98:462–467.

Yu,J.&vandenEngh,G.(1995)Flow-sortandgrowthofsinglebacterialcellstransformedwithcosmidandplasmidvectorsthatincludethegeneforgreen-fluorescentproteinasavisiblemarker.Abstractsofpaperspresentedatthe1995meetingon“GenomeMappingandSequencing”,ColdSpringHarbor.p.293.

Clontechniques references (inchronologicalorder):

LivingColorsRedFluourescentProtein.(October1999)Clontechniques xIV(4):2–6.

LivingColorsFluorescentTimer.(April2001)CLONTECHniquesxVI(2):14–15.

LivingColorsDsRed2.(July2001)Clontechniques xVI(3):2–3.

LivingColorsHcRed.(April2002)Clontechniques xVII(2):12–13.

LivingColorsDsRed-Express(July2002)ClontechniquesxVII(3):16–17.

DestabilizedDsRed-ExpressandHcRedVectors(October2002)Clontechniques xVII(4).

Reefcoralfluorescentproteins(July2003)Clontechniques xVIII(3):6–7.

VII. References continued



ForacompletelistingofallClontechproducts,pleasevisitwww.clontech.com

Product Cat. No.

• RCFPExpressionVectors many• EnhancedGFPExpressionVectors many

• CalPhos™MammalianTransfectionKit 631312

• CLONfectin™TransfectionReagent 631301

• Adeno-X™ExpressionSystem1 631513

• Adeno-X™ExpressionSystem2 many withCreator™Technology

• Adeno-XDsRed2MarkerVirus 632417

• Adeno-XEGFPMarkerVirus 630115

• Retro-X™System 631508

VIII. Related Products

Protocol No. PT3404-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR37085 ��


Notes



Notes

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