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Living Colors®
User Manual
PT2040-1 (PR1Y691)Published 26 November 2001
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Clontech Laboratories, Inc. www.clontech.com Protocol No. PT2040-1 2 Version No. PR1Y691
Living Colors® User Manual
I. Introduction 3
II. Properties of GFP and GFP Variants 5
III. Expression of GFP and GFP Variants 14
IV. Detection of GFP, GFP Variants, and DsRed 19
V.Purified Recombinant GFP and GFP Variants 28
VI. GFP Antibodies 30
VII. Troubleshooting Guide 32
VIII. References 36
IX. Related Products 42
Appendix: Recommended Resources & Reading 46
List of Figures
Figure1. GenealogyofGFPproteins. 4
Figure2. AminoacidsequencedifferencesbetweenwtGFP anditsvariants. 5
Figure3. ExcitationandemissionspectraofwtGFPanda red-shiftedGFPexcitationvariant(GFP-S65T). 6
Figure4. DsRed,EGFP,ECFP,andEYFPhavedistinctabsorptionandemissionspectra. 7
Figure5. Flowcytometryanalysisofd2EGFPstability. 9
Figure6. ComparisonoffluorescenceintensityofGFPinsonicationbufferversusGFPincelllysates. 25
Figure7. Relativefluorescenceintensityoftwo-foldserialdilutionsofasuspensionofGFP-transformedcellsinsonicationbuffer. 27
List of Tables
TableI. LivingColors®FluorescentProteins:SpectralProperties 13
TableII. PartialListofProteinsExpressedasFusionstoGFP 15–16
TableIII. MulticoloranalysiswithLivingColors®Proteins 21
Table of Contents
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Thebioluminescent jellyfishAequoreavictoriaproduces lightwhenenergy istransferredfromCa2+-activatedphotoproteinaequorintogreenfluorescentpro-tein(GFP;Shimomuraetal.,1962;Morin&Hastings,1971;Wardetal.,1980).Thecloningofthewild-typeGFPgene(wtGFP;Prasheretal.,1992;Inouye&Tsuji,1994a)anditssubsequentexpressioninheterologoussystems(Chalfieetal.,1994;Inouye&Tsuji,1994a;Wang&Hazelrigg,1994)establishedGFPas a novel genetic reporter system. When expressed in either eukaryotic orprokaryoticcellsandilluminatedbyblueorUVlight,GFPyieldsabrightgreenfluorescence.Light-stimulatedGFPfluorescence is species-independentanddoesnotrequireanycofactors,substrates,oradditionalgeneproductsfromA.victoria.Additionally,detectionofGFPanditsvariantscanbeperformedinlivingcellsandtissuesaswellasfixedsamples.
ClontechmakesGFPandGFPvariantsavailableforresearchpurposesthroughitslineofLiving Colors®Fluorescent Proteins (Figure1).LivingColorsproteinsareexcellentreportersforgeneexpressionandproteinlocalizationstudies.Fol-lowingisalistoftheLivingColorsGFPvariantscurrentlyoffered.Youwillfindthatthesevariantsarewellsuitedforfluorescencemicroscopyandflowcytometry.
•GFPexcitationvariants: – EGFP, enhanced green fluorescent protein (GFPmut1; Cormack et
al.,1996),anddestabilized EGFP (dEGFP;Lietal.,1998)havered-shiftedexcitationspectra.Whenexcitedwithblue light, theyfluoresce4–35-foldmorebrightlythanwtGFP.
•GFPemissionvariants: – EYFP (enhanced yellow fluorescent protein) contains four amino acid
substitutionsthatshifttheemissionfromgreentoyellowish-green.ThefluorescencelevelofEYFPisroughlyequivalenttothatofEGFP.
– ECFP(enhancedcyanfluorescentprotein)containssixaminoacidsub-stitutions,oneofwhichshiftstheemissionspectrumfromgreentocyan(Heim&Tsien,1996;Miyawakietal.,1997).Itisasuitabledonormoleculeinfluorescenceresonanceenergy transfer(FRET)studiesandcanbeusedfordual-labelingfluorescencemicroscopyinconjunctionwithEYFP(Greenetal.,2000).
•UV-OptimizedGFPvariant: – GFPuvisreportedtobe18timesbrighterthanwtGFPwhenexpressed
inE.coliandexcitedbystandardUVlight(Cramerietal.,1996).ThisvariantcontainsadditionalaminoacidmutationswhichalsoincreasethetranslationalefficiencyoftheproteininE.coli.
I. Introduction
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•RedFluorescentProtein: – WealsoofferDsRed2,aredfluorescentproteinthatderivesfromacoral
oftheDiscosomagenus,referredtoasDiscosomasp.(Ds).Becauseofitsdistinctredemission,DsRed2isaperfectcomplementtothegreen,yellow,andcyanfluorophores.WiththedevelopmentoffiltersdesignedspecificallyfordetectingEGFP,EYFP,ECFP,andDsRed,it’snowpos-sibletousethesereportersincombinationtocarryouttwo-,three-,or,even,four-coloranalyses.TheRedFluorescentProtein,DsRed,ismorecarefullydescribedinaseparateUserManual,PT3404-1,whichcanbedownloadedfromourwebsiteatwww.clontech.com.
Inadditiontotheirchromophoremutations,theenhancedGFPvariantgenesEGFP,EYFP,andECFPcontainmorethan190silentmutationsthatcreateanopenreadingframecomprisedalmostentirelyofpreferredhumancodons(Haas,etal.,1996).Furthermore,upstreamsequencesflankingthecodingregionshavebeenconvertedtoaKozakconsensustranslationinitiationsite(Kozak,1987),andpotentiallyinhibitoryflankingsequencesintheoriginalcDNAclones(lgfp10&pGFP10.1;Chalfieetal.,1994)wereremoved.AllofthesechangesenhancethetranslationalefficiencyofthemRNA—and,consequently,theexpressionoftheGFPvariant—inmammalianandplantsystems.
I. Introduction continued
Figure 1. Genealogy of GFP proteins. GFPmut1(Cormacketal.,1996),thered-shiftedvariantusedtocreatethevariantsshown,isnotsoldbyClontech.
GFPuvGFPmut1
GFP (wt)
EGFP
d2EGFPd1EGFP d4EGFPEYFP ECFP
d2EYFP d2ECFP
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II. Properties of GFP and GFP Variants
A. The GFP Chromophore: Structure and Biosynthesis TheGFPchromophoreconsistsofacyclic tripeptidederived fromSer-
Tyr-Gly(aminoacids65–67)intheprimaryproteinsequence(Codyetal.,1993)andisonlyfluorescentwhenembeddedwithinthecompleteGFPprotein.Ofthecomplete238-amino-acidpolypeptide,aminoacids7–229arerequiredforfluorescence(Lietal.,1997).ThecrystalstructureofGFPhasrevealedatightlypackedβ-canenclosinganα-helixcontainingthechro-mophore(Ormöetal.,1996;Yang,F.etal.,1996).Thisstructureprovidestheproperenvironmentforthechromophoretofluoresce.NascentGFPisnotfluorescent,sincechromophoreformationoccurspost-translationally.Thechromophoreisformedbyacyclizationreactionandanoxidationstepthatrequiresmolecularoxygen(Heimetal.,1994;Davis,D.F.etal.,1995).Thesestepsareeitherautocatalyticorusefactorsthatareubiquitous,sincefluorescentGFPformsinabroadrangeoforganisms.Chromophoreforma-tionmaybetherate-limitingstepformaturationofthefluorescentprotein(Heimetal.,1994;Davis,D.F.etal.,1995).Wild-typeGFPabsorbsUVandbluelightandemitsgreenlight.
B. GFP Excitation Variants Thered-shiftedvariantsEGFPanddEGFP(destabilizedforrapidturnover)
containmutationsinthechromophore(Figure2)whichshiftthemaximalexcitationpeakto~490nm(Figure3).
wt GFP:
EGFP:
EYFP:
ECFP:
Phe 64 Ser Tyr Gly Val Gln 69 . . . Ser72 . . .Tyr 145 Asn146. . . Met153. . . Val163. . . Thr203
Leu 64 Thr Tyr Gly Val Gln 69
Phe64 Gly Tyr Gly Leu Gln69 . . . Ala72. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .Tyr203
Leu 64 Thr Trp Gly Val Gln 69. . . . . . . . . . . . . . . . . Ile146. . . . Thr153. . . . Ala163
Figure 2. Amino acid sequence differences between wt GFP and its variants. Aminoacidsub-stitutionsareunderlined.
The major excitation peak of the red-shifted variants encompasses theexcitationwavelengthofcommonlyusedfiltersets.Similarly,theargon-ionlaserused inmostFACSmachinesandconfocalscanning lasermicro-scopesemitsat488nm,soexcitationofthered-shiftedGFPvariantsismuchmoreefficientthanexcitationofwtGFP.Inpracticalterms,thismeansthedetectionlimitsinbothmicroscopyandFACSareconsiderablylowerwiththered-shiftedvariants.Dual-labelingexperimentscanbeperformedbyselectiveexcitationofwtGFPandred-shiftedGFP(Kainetal.,1995;Yang,T.T.etal.,1996b).ItisalsopossibletouseEGFPandGFPuvindouble-labelingexperiments.
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II. Properties of GFP and GFP Variants continued
• EGFP contains two amino acid substitutions: F64L and S65T(Figure2).Basedonspectralanalysis,EGFPfluoresces35-foldmoreintenselythanwtGFPwhenexcitedat488nm(Cormacketal.,1996;Yang,T.T.etal.,1996a)duetoanincreaseinitsextinctioncoefficient(Em;TableI)andahigherefficiencyofchromophoreformation.EGFPchromophoreformationismuchmoreefficientat37°CthanwtGFP;95% of the soluble EGFP protein contains the chromophore whenexpressedatthistemperature(Pattersonetal.,1997).
C. GFP Emission Variants • Cyan Emission Variant (ECFP) TheECFPvariantcontainssixaminoacidsubstitutions(Figure2).One
of these,Tyr-66 toTrp, shifts thechromophore’sexcitationmaximato433nm(majorpeak)and453nm(minorpeak),andtheemissionmaximato475nmwithasmallshoulderat501nm(TableI;Figure4).TheotherfivesubstitutionsenhancethebrightnessandsolubilityoftheproteininamannersimilartotheotherEGFPvariants.
• Yellow Emission Variant (EYFP) The EYFP variant contains four different amino acid substitutions
(Figure2)thatshiftthefluorescencefromgreen(509nm)toyellow-green(527nm;TableI;Figure4).AlthoughEYFP’sfluorescenceexcitationmaximumis513nm,itcanbeefficientlyexcitedat488nm,thestandardlaserlineforanargon-ionlaser.
Figure 3. Excitation and emission spectra of wt GFP and a red-shifted GFP excitation variant (GFP-S65T).Excitation:dashedlines.Emission:solidlines.TheemissiondataforwtGFPwereob-tainedbyexcitingtheproteinat475nm.(TheemissionpeakforwtGFPisseveral-foldhigherwhenilluminatedat395nm).TheemissiondataforGFP-S65Twereobtainedbyexcitingtheproteinat489nm(Heimetal.,1995).EGFPandGFP-S65Thavesimilarspectra.
400 500Wavelength (nm)
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II. Properties of GFP and GFP Variants continued
Figure 4. DsRed, EGFP, ECFP, and EYFP have distinct absorption and emission spectra. Panel A.Absorbance.Panel B.Emission.Curvesarenormalized;theheightofeachcurveisnotanindicationofrelativesignalstrengthperfluorophore.EGFP,ECFP,andEYFPareallvariantsofthegreenfluorescentproteinfromAequoreavictoria,denotedasAvinthefigure.DsRedisderivedfromacoraloftheDiscosomagenusreferredtoasDiscosomasp.(Ds).(SpectraforEGFP,ECFP,andEYFPwereprovidedbyD.W.Piston,VanderbiltUniversity.)
A
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II. Properties of GFP and GFP Variants continued
D. UV-Optimized GFP Variant GFPuv isoptimized formaximalfluorescencewhenexcitedbyUV light
(360–400 nm) and for higher bacterial expression. The GFPuv variantis idealforexperimentsinwhichGFPexpressionwillbedetectedusingUVlightforchromophoreexcitation(e.g.,forvisualizingbacteriaoryeastcolonies).GFPuvwasdevelopedbyCramerietal.(1996).
GFPuvcontainsthreeaminoacidsubstitutions(Phe-99toSer,Met-153toThr,andVal-163toAla[basedontheaminoacidnumberingofwtGFP]),noneofwhichalter thechromophoresequence.ThesemutationsmakeE.coliexpressingGFPuvfluoresce18timesbrighterthanwtGFP(Cramerietal.,1996).WhilethesemutationsdramaticallyincreasethefluorescenceofGFPuvthroughtheireffectsonproteinfoldingandchromophoreformation,theemissionandexcitationmaximaremainatthesamewavelengthsasthoseofwtGFP(Cramerietal.,1996;Figure3;TableI).However,GFPuvhasagreaterpropensitytodimerizethanwtGFP.
GFPuvexpressedinE.coli isasoluble,fluorescentproteinevenunderconditionsinwhichthemajorityofwtGFPisexpressedinanonfluorescentformininclusionbodies.ThisGFPvariantalsoappearstohavelowertoxicitythanwtGFP;hence,theE.colicontainingGFPuvgrowtwotothreetimesfasterthanthoseexpressingwtGFP(Cramerietal.,1996).Furthermore,theGFPuvgene isasyntheticGFPgene inwhichfive rarelyusedArgcodons from thewtgenewere replacedbycodonspreferred inE.coli.Consequently,theGFPuvgeneisexpressedveryefficientlyinE.coli.
E. Destabilized GFP Variants Inmammaliancells,destabilizedvariants(dEGFP,dECFP,anddEYFP)aredegradedmorerapidlythanwtGFP.Inordertocreatethedestabilizedvari-ants,EGFP,ECFP,andEYFPwerefusedtoaminoacidresidues422–461ofmouseornithinedecarboxylase(MODC).ThisC-terminalregionofMODCcontainsaPEST(Pro-Glu-Ser-Thr)aminoacidsequencethattargetstheproteinforrapidintracellulardegradation.Thed1,d2,andd4variantsex-hibithalf-livesof1,2,or4hours,respectively,comparedtoEGFP,ECFP,andEYFP,whichhavehalf-livesof>24hours.Half-livesweredeterminedbyflowcytometry(Figure5),fluorescencemicroscopy,andWesternblotanalysis(Lietal.,1998;July1999&April1998Clontechniques).
Thefluorescenceintensityandspectralpropertiesofdestabilizedvariantsare identical to thoseof theoriginal chromophore,butbecauseof theirshortenedhalf-lives,destabilizedvariantscanbeusedtomoreaccuratelymeasurethekineticsofpromoteractivityorthetransientexpressionofaproteintowhichtheyarefused.
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II. Properties of GFP and GFP Variants continued
Time after CHX addition (hr)
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Figure 5. Flow cytometry analysis of d2EGFP stability. CHO-K1Tet-Off™cellsweretransfectedwithpTRE-EGFPorpTRE-d2EGFP.After24hr,thetransfectedcellsweretreatedwith100µg/mlcycloheximide(CHX)for0,1,2,or3hrtoinhibitproteinsynthesis.TheCHX-treatedcellswerehar-vestedwithEDTA,and1x105cellswereusedforflowcytometryanalysis.Thepercentagesofcellswithfluorescenceabovebackgroundareplottedonthey-axis.Valuesarenormalizedtofluorescencelevelsatthezerotimepoint,definedas100%.After3hr,fluorescenceintensityofthecellpopulationexpressingd2EGFPwasreducedto30%ofinitialintensity.
F. Acquisition and Stability of GFP Fluorescence 1. Protein solubility Inbacterialexpressionsystems,muchoftheexpressedwtGFPisfound
inan insoluble,nonfluorescent formin inclusionbodies. Incontrast,almostallEGFPandGFPuv isexpressedasasoluble,fluorescentprotein.
2. Photobleaching GFPfluorescenceisverystableinafluorometer(Ward,pers.comm.).
Even under the high-intensity illumination of a fluorescence micro-scope,GFP ismore resistant tophotobleaching than is fluorescein(Wang&Hazelrigg,1994;Niswenderetal.,1995).ThefluorescenceofwtGFPandEGFPisquitestablewhenilluminatedwith450–490nmlight(themajorexcitationpeakforthered-shiftedexcitationvariants,buttheminorpeakforwtGFP).SomephotobleachingoccurswhenwtGFPisilluminatednearitsmajorexcitationpeakwith340–390nmor395–440nmlight(Chalfieetal.,1994;Niswenderetal.,1995).TherateofphotobleachingofwtGFPand itsgreenvariantsalsovarieswith theorganismbeing studied; for example,GFPfluorescence isquitestableinDrosophila(Wang&Hazelrigg,1994)andzebrafish.InC.elegans,10mMNaN3acceleratesphotobleaching(Chalfieetal.,1994).StudiesofthephotobleachingcharacteristicsofGFPuvhavenotbeenperformedtodate.
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II. Properties of GFP and GFP Variants continued
WhenusingECFP,photobleachingdoesnotposeamajorproblem,andwerecommendusingthisvariantifphotobleachinginterfereswith your studies. (Rapid photobleaching has been a problemwithpreviouslydescribedbluevariantsofGFP.)However,ifyoudecidetofixandmountyourspecimens,commerciallyavailablemountingsolutionsthatdonotcontainantibleachingreagentscancauseseverephotobleachingwithinsecondswhentheslidesareexposedtolightduringfluorescencemicroscopy.Besuretokeepmountedslidesinthedarkat4°C.
3. Stability to oxidation/reduction GFPneedstobeinanoxidizedstatetofluorescebecausechromo-
phoreformationisdependentuponanoxidationofTyr-66(Heimetal.,1994).Strongreducingagents,suchas5mMNa2S2O4or2mMFeSO4,convertGFPintoanonfluorescentform,butfluorescenceisfullyrecoveredafterexposuretoatmosphericoxygen(Inouye&Tsuji,1994b).Weakerreducingagents,suchas2%β-mercaptoethanol,10mMdithiothreitol(DTT),10mMreducedglutathione,or10mML-cysteine(Inouye&Tsuji,1994b),ormoderateoxidizingagents(Ward,pers.comm.),donotaffectthefluorescenceofGFP.
4. Stability to pH wtGFP retains fluorescence in the rangepH5.5–12; however,
fluorescenceintensitydecreasesbetweenpH5.5andpH4,anddropssharplyabovepH12(Bokman&Ward,1981).EGFPexhib-itsareducedrangeofpHstability.Forthisvariant,fluorescenceisstablebetweenpH7.0andpH11.5,dropssharplyabovepH11.5,anddecreasesbetweenpH7.0andpH4.5,retainingabout50%offluorescenceatpH6.0(Pattersonetal.,1997;Ward,pers.comm.).BecauseGFPissosensitivetopH,itcanbeusedasanintracellularpHindicatorinlivingcells(Kneenetal.,1998).
5.Stability to chemical reagents GFPretainsitsfluorescenceinmilddenaturants,suchas1%SDS
or8Murea,andafterfixationwithglutaraldehydeorformaldehyde.FullydenaturedGFPisnotfluorescent.GFPissensitivetosomenailpolishesusedtosealcoverslips(Chalfieetal.,1994;Wang&Hazelrigg,1994);therefore,usemoltenagaroseorrubberce-menttosealcoverslipsonmicroscopeslides.Fluorescenceisalsoquenchedbythenematodeanestheticphenoxypropanol(Chalfieetal., 1994).GFPfluorescence is irreversiblydestroyedby1%H2O2andsulfhydryl reagentssuchas1mMDTNB (5,5'-dithio-bis-[2-nitrobenzoicacid]) (Inouye&Tsuji,1994b).Manyorganicsolventscanbeusedatmoderateconcentrationswithoutabolishingfluorescence;however,theabsorptionmaximummayshift.
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II. Properties of GFP and GFP Variants continued
GFPisnonfluorescentinabsoluteethanolandisunlikelytowithstandthecompletedehydrationrequiredforparaffinorplasticembedding.
6. Protein stability in vitro: GFP is exceptionally resistant to heat (Tm=70°C), alkaline pH,
detergents,chaotropicsalts,organicsolvents,andmostcommonpro-teases,exceptpronase(Bokman&Ward,1981;Ward,1981).SomeGFPfluorescencecanbeobservedwhennanogramamountsofproteinareresolvedonnativeor1%SDSpolyacrylamidegels(Inouye&Tsuji,1994a).
FluorescenceislostifGFPisdenaturedbyhightemperature,pHex-tremes,orguanidiniumchloride,butcanbepartiallyrecoverediftheproteinisallowedtorenature(Bokman&Ward,1981;Ward&Bokman,1982).Athiolcompoundmaybenecessarytorenaturetheproteinintothefluorescentform(Surpin&Ward,1989).
in vivo: GFPappearstobestablewhenexpressedinvariousorganisms;EGFP
hasanestimatedhalf-lifeof>24hours(Lietal.,1998). 7. Temperature sensitivity of GFP chromophore formation AlthoughfluorescentGFPishighly thermostable(seeabove), itap-
pears that the formation of the GFP chromophore is temperaturesensitive. Inyeast,GFPfluorescencewasstrongestwhen thecellsweregrownat15°C,decreasingtoabout25%ofthisvalueasthein-cubationtemperaturewasraisedto37°C(Limetal.,1995).However,GFPandGFP-fusionproteins synthesized inS. cerevisiaeat 23°Cretain fluorescence despite a later shift to 35°C (Lim et al., 1995).It has also been noted that E. coli expressing GFP show strongerfluorescencewhengrownat24°Cor30°Ccomparedto37°C(Heimetal.,1994;Ward,pers.comm.).MammaliancellsexpressingGFPhavealsobeenseentoexhibitstrongerfluorescencewhengrownat30–33°Ccomparedto37°C(Pines,1995;Ogawaetal.,1995).
AlltheenhancedGFPvariantsshowlittledifferenceinfluorescencewhenexpressedateither25°Cor37°C.Themutationsthatincreasetheefficiencyofproteinfoldingandchromophoreformation(Cormacketal.,1996,Cramerietal.,1996)alsosuppressthethermosensitivityofchromophoreformation.
8. Dimerization of GFP GFPisfluorescenteitherasamonomerorasadimer.Theratioof
monomericanddimericformsdependsontheproteinconcentrationandenvironment.wtGFPdimerizesviahydrophobic interactionsatproteinconcentrationsabove5–10mg/mlandinhigh-saltconditions.
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Dimerizationresultsina4-foldreductionintheabsorbanceat470nmanda concomitant increase inabsorbanceat 395nm (Ward, pers.comm.).GFPuvhasagreaterpropensitytodimerizethanwtGFP;the470-nmexcitationpeakissuppressedatproteinconcentrationsabout5-foldlowerthanforwtGFP(Ward,pers.comm.).InmostexperimentalsystemsusingwtGFPasareporter,themonomericformwillpredomi-nate;however,whenGFPuvisusedasthereporter,dimerizationmaybeevidentevenatmoderateexpressionlevels.
G. Sensitivity and fluorescence intensity 1. General considerations GFPfusionproteinshavebeenfoundtogivegreatersensitivityand
resolutionthanstainingwithfluorescentlylabeledantibodies(Wang&Hazelrigg,1994).GFPfusionsaremoreresistanttophotobleachingandexhibitlowbackgroundfluorescence(Wang&Hazelrigg,1994).
Because GFPuv, EGFP, and dEGFP exhibit higher extinction coef-ficients thanwtGFP, theyprovide significantly increased sensitivityasreportermolecules.Furthermore,subcellularlocalizationofEGFP(e.g.,tothecytoskeleton)canproduceanevenmoreintensesignalbyconcentratingthesignaltoaspecificareawithinthecell.However,forsomeapplications,thesensitivityofGFPmaybelimitedbyautofluo-rescenceorlimitedpenetrationoflight.StudieswithwtGFPexpressedinHeLacells(Niswenderetal.,1995)haveshownthatthecytoplasmicconcentrationmustbegreaterthan~1.0µMtoobtainasignalthatistwicetheautofluorescence.Thisthresholdfordetectionislowerwiththe red-shifted excitation variant: ~100 nM for EGFP (Piston, pers.comm.).
2. As a quantitative reporter Thesignal fromGFPand itsvariantsdoesnothaveanyenzymatic
amplification;hence,thesensitivityofGFPwillprobablybelowerthanthatforenzymaticreporterssuchasβ-galactosidase,SEAP,andfireflyluciferase.However,GFPsignalscanbequantifiedbyflowcytometry,confocalscanninglasermicroscopy,andfluorometricassays.PurifiedGFPandGFPvariantscanbequantifiedinafluorometer-basedassayinthelow-nanogramrange.Furthermore,usingdestabilizedGFPsasreporterscanincreasesensitivitybypreventingaccumulationofGFPunderbasalornon-inducedexpression.Therefore,destabilizedGFPseffectivelyextendtheinductionwindowbyincreasingfold-induction.
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TABLE I. LIVInG COLORS® FLUORESCEnT PROTEInS: SPECTRAL PROPERTIES
Protein Excitation Emission Em (cm–1M–1) Quantum Yield (%) Maxima (nm) Maxima (nm)
wtGFP 395(470) 509(540) 9,500a ~80a
EGFPd 488 507 55,000b ~60b
ECFPd 433(453) 475(501) 26,000b ~40b
EYFPd,e 513 527 84,000b ~61b
GFPuv 395 509
DsRede 558 583 75,000c ~70c
aPattersonetal.,1997bDavePiston,pers.comm.c Bairdetal.,2000;SlightlylowervaluesarereportedbyPattersonetal.,2001.dThedestabilizedvariant(dEGFP,dECFP,ordEYFP)hasidenticalexcitationandemission
maxima,butashorterhalf-life.eEYFPandDsRedcanalsobeexcitedat488nm.SeeFigure4.
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A. Suitable Host Organisms and Cells GFP fluorescence is species-independent. Fluorescence has been
reportedfrommanydifferenttypesofGFP-expressinghosts.BelowisapartiallistingoforganismsthatcanexpressfluorescentGFP;amoreex-tensivelistingwithreferencescanbefoundatwww.clontech.com/gfp.
1. Microbes Agrobacteriumtumefaciens Myxococcusxanthus Anabaena Polysphondyliumpallidum Bacillussubtilis Pseudomonas Bartonellahenselae Rhizobiummeloliti Candidaalbicans Saccharomycescerevisiae Caulobactercrescentus Salmonella Dictyostelium Schizosaccharomycespombe Escherichiacoli Ustilagomaydis Helicobacterpylori Yersinia Mycobacterium
2. Invertebrates Aedesaegypti Caenorhabditiselegans Diamondbackmoth Drosophilamelanogaster Lytechinuspictus
3. Vertebrates Fish, avian,andmammaliancell lines (specific cell lines listedon
web) Mouse Xenopus Zebrafish
4. Plants Arabidopsisthaliana(thalecress) Citrussinensis(L.)Osbeckcv.Hamlin(anembryogenicsweet-orange
cellline,H89) Glycinemax(soybean) Hordeumvulgar(barley) Nicotianabenthamiana&Nicotianaclevelandii(tobacco) Onion Zeamays(maize)
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B. Expression of GFP Fusion Proteins GFPhasbeenexpressedasafusiontomanydifferentproteins(TableII).
Inmanycases,chimericgenesencodingeitherN-orC-terminalfusionstoGFPretainthenormalbiologicalactivityoftheheterologouspartner,aswellasmaintainingfluorescentpropertiessimilartonativeGFP(Flachetal.,1994;Wang&Hazelrigg,1994;Marshalletal.,1995;Stearns,1995).TheuseofGFPanditsvariantsinthiscapacityprovidesa“fluorescenttag”ontheprotein,whichallowsforinvivolocalizationofthefusionprotein.GFPfusionscanprovideenhancedsensitivityandresolutionincomparisontostandardantibodystainingtechniques(Wang&Hazelrigg,1994),andtheGFPtageliminatestheneedforfixation,cellpermeabilization,andanti-bodyincubationstepsnormallyrequiredwhenusingantibodiestaggedwithchemicalfluorophores.Lastly,useoftheGFPtagpermitskineticstudiesofproteinlocalizationandtrafficking(Flachetal.,1994;Wang&Hazelrigg,1994).
TABLE II. PARTIAL LIST OF PROTEInS EXPRESSED AS FUSIOnS TO GFP
Host Cell orProtein Organism Localization References
CotE Bacillussubtilis Forespore Webbetal.,1995
DacF,SpoIVA Bacillussubtilis Prespore/mothercell Lewis&Errington,1996
Coronin D.discoideum Phagocyticcups Maniaketal.,1995
Myosin D.discoideum Mooresetal.,1996
YopEcytotoxin Yersinia Jacobietal.,1995
HistoneH2B Yeast Nucleus Flachetal.,1994; Schlenstedtetal.,1995
Tubulin Yeast Microtubules Stearns,1995
Nuf2p Yeast Spindle-polebody Silver,1995;Kahanaetal.,1995
Mitochondrialmatrix Yeast Mitochondria Cox,1995 targetingsignal
Npl3p Yeast Nucleus Corbettetal.,1995
Nucleoplasmin Yeast Nucleus Limetal.,1995
Cap2p Yeast Waddleetal.,1996
Swi6p Yeast Sidorvaetal.,1995
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TABLE II continued. PARTIAL LIST OF PROTEInS EXPRESSED AS FUSIOnS TO GFP
Host Cell orProtein Organism Localization References
HMG-R Yeast Hamptonetal.,1996
Bud10p Yeast Halmeetal.,1996
Pmp47 Yeast Peroxisome Dyeretal.,1996
Act1p,Sac6p,Abp1pYeast Cytoskeleton Doyleetal.,1996
p93dis1 Fissionyeast Spindle-polebody& microtubules Nabeshimaetal.,1995
Exu Drosophila Oocytes Wang&Hazelrigg,1994
Mei-S332 Drosophila Centromere Kerrebrocketal.,1995
Tau Drosophila Microtubules Brand,1995
β-galactosidase Drosophila cellmovement Shigaetal.,1996
Streptavidin Insectcells Oker-Blometal.,1996
ObTMVmovement protein Tobacco Filaments Beachy,1995; Heinleinetal.,1995
PotatovirusX coatprotein Potatoplant Viralinfection SantaCruzetal.,1996
GST Zebrafish Cytoplasmic(?) Petersetal.,1995
11β-HSD2 Mammaliancells Endoplasmicreticulum Náray-Fejes-Tóthetal.,1996
MAP4 Mammaliancells Microtubules Olsonetal.,1995
Mitochondrial targetingsignal Mammaliancells Mitochondria Rizzutoetal.,1995
Cyclins Mammaliancells Nucleus,micro- tubules,orvesicles Pines,1995
PML Mammaliancells Ogawaetal.,1995
Humanglucocor- Mammaliancells Ogawaetal.,1995 ticoidreceptor Careyetal.,1996
Ratglucocorticoid Mammaliancells Rizzutoetal.,1995 receptor Htunetal.,1996
ChromograninB HeLacells Secreted Kaetheretal.,1995
HIVp17protein HeLacells Nucleus Bianetal.,1995
NMDAR1 HEK293cells Membrane Marshalletal.,1995
CENP-B Humancells Nucleus Sullivanetal.,1995
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C. GFP Expression in Mammalian Cells Appropriatevectorsmaybetransfectedintomammaliancellsbyavariety
oftechniques,includingthoseusingcalciumphosphate(Chen&Okayama,1988),DEAE-dextran (Rosenthal,1987),various liposome-based trans-fectionreagents(Sambrooketal.,1989),andelectroporation(Ausubeletal.,1994).Anycalciumphosphatetransfectionproceduremaybeused,but we recommend using the CalPhosTM Mammalian Transfection Kit(#K2051-1)forhighcalciumphosphate-mediatedtransfectionefficiencieswithan incubationofonly2–3hours.Likewise,any liposome-mediatedtransfectionproceduremaybeused,butwerecommendusingCLONfectinTM(#8020-1)forhigh,reproducibletransfectionefficienciesinavarietyofcelltypes.Forfurtherinformationoncellculturetechniques,werecommendCultureofAnimalCells,ThirdEdition,R.I.Freshney(1993,Wiley-Liss).
The efficiency of a mammalian transfection procedure is primarilydependentonthehostcellline.Therefore,whenworkingwithacelllineforthefirsttime,itisadvisabletocomparetheefficienciesofseveraltransfec-tionprotocols.ThiscanbestbeaccomplishedusingoneoftheGFPvectorswhichhastheCMVimmediateearlypromoterforhigh-levelexpressioninmostcelllines.
In most cases, GFP expression may be detected by fluorescencemicroscopy,FACSanalysis,orfluorometerassays24–72hourspost-trans-fection,dependingonthehostcelllineused.Ifyouusedelectroporation,waituntil48hourspost-transfectiontoassayorbeginselectiontoallowcells to recover from the electroporation procedure. To visualize GFP-expressingcellsbyfluorescencemicroscopy,growthecellsonasterileglasscoverslipplacedina60-mmcultureplate.Alternatively,aninvertedfluorescencemicroscopemaybeusedfordirectobservationoffluorescentcellsinthecultureplate.
D. GFP Expression in Plants
InArabidopsis,werecommendusingEGFPinsteadofwtGFP,becausethewtcodingsequencecontainsaregionrecognizedinArabidopsisasacrypticplantintron(bases400–483).Thisintronissplicedout,resultinginanonfunctionalprotein(Haseloff&Amos,1995).HaseloffandAmosreportsuccessfulexpressioninArabidopsisofamodifiedversionofGFPinwhichthecrypticintronsequenceshavebeenaltered.ThecrypticintronhasalsobeenremovedbythechangestocodonusageinEGFP,ECFP,andEYFP.A modified GFP gene with the same codon usage as EGFP has beenusedtoexpressGFPinmanyplantsystemsusingavarietyoftransfectiontechniques (Chiuet al., 1996).FunctionalwtGFPhasbeen transientlyexpressedinseveralotherplantspecies,indicatingthatthecrypticintronisunrecognizableinotherplantspecies.
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E. GFP Expression in YeastwtGFPandGFPuvallexpresswellinyeast.Forexpressioninyeast,theGFPvariantcontainedinClontech’svectorsmustfirstbeisolated(byPCRorbyexcision from theplasmid)and inserted intoanappropriateyeastexpressionvector.Note thatGFPgeneswithhumancodonusage(i.e.,enhancedvariantssuchasEGFP)arenotefficientlyexpressedinyeast(Cormacketal.,1997).Alsonote thatS.cerevisiaestrainscarrying theade2mitochondrialmutationaccumulatearedpigment,whichgivesthecellsanautofluorescencethatinterfereswithdetectionofGFP(Smirnovetal.,1967;Weismanetal.,1987).OurYeastProtocolsHandbook(PT3024-1)containsadditionalinformationonyeastgrowthandmaintenance,preparationofyeastcompetentcells,andtransformationofyeastcells.Toobtainacopy,pleasevisitourwebsiteatwww.clontech.com.Additionally,werecommendGuidetoYeastGeneticsandMolecularBiol-ogy,C.GuthrieandG.R.Finkeds.(Vol.194inMethodsinEnzymology,1991,AcademicPress).
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A. Microscopy 1. Conventional microscopy a. Filter sets Althoughyoucanachieveexcellentresultsusingstandardfiltersets,
suchasFITCfilterstodetectEGFP,andrhodamineorpropidiumiodidefilterstodetectDsRed,optimizedfiltersetsfordetectingGFP,GFPvariants,andDsRedhavebeendevelopedbyOmegaOpticalInc.andChromaTechnologyCorp.Ontheirwebsites(www.omega-filters.com;www.chroma.com),youwillfinddetailedinformationabouthow todetectLivingColors®FluorescentProteins.Opticalproperties,recommendeduses,andsampledataareallcarefullydescribedsothatyoucanchoosethefiltersthatbestsuityourneeds.With researchers increasingly interested in multi-color analysis,eachcompanyhasdoneextensivetestingtofindtheoptimalfiltercombinationsforseparatingcyan,green,yellow,andredfluores-cence.Youcanreadaboutthetestresults,includingsuggestionsforexperimentaldesignanddatacollection,andviewcolorimagesbyvisitingeachofthesewebsites.
Ifyoushouldusefluoresceinisothiocyanate(FITC)filters,keepinmindthatthesefiltersetsusuallytransmitexcitationlightat450–500nm—wellabovethemajorexcitationpeak(395nm)ofwtGFP.Thesefilters,therefore,produceafluorescentsignalfromwtGFPthat is several-fold less than would be obtained with filters thattransmitat395nm.However,itisnotadvisabletoexciteat395nmbecauseatthiswavelength,GFPrapidlyphotoisomerizesandlosesthefluorescentsignal.
IncontrasttowtGFP,EGFPhasasingle,strong,red-shiftedexcitationpeakat488nm,makingitwellsuitedfordetectionincommonlyusedequipmentwhichutilizeFITCoptics.Becauselivingcellstoleratelongerwavelengthsbetterduetothelowerenergies,thefluorescentsignalobtainedbyilluminatingEGFPat~488nmismorestableandlesstoxicthanthatofwtGFP.
b. Light source Theemissionintensityfromalightsourcecanvarywithwavelength.
Becausetheintensityoftheexcitationlightdirectlyaffectsthebright-nessofareporter,itisimportanttocomparetheexcitationspectraofyourchosenreporterswiththeemissionspectrumofthelightsource.Conventionalfluorescencemicroscopesareequippedwitheitheramercury-orxenon-arc lamp.Both lampsaresuitable forexcitingfluorescentproteins.Butbeawarethat,whereasthelightintensityfromaxenonlampdeviatesverylittleacrosstheblue,green,yel-low,andredregions,theemissionfromamercurylamphassharppeaksinthecyanandredregions.Therefore,fluorophoresexcited
IV. Detection of GFP, GFP Variants, and DsRed
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IV. Detection of GFP, GFP Variants, and DsRed continued
bycyanandredlightmayappearbrighterwhenexcitedbyamercurylamp.Checkthemanufacturers’specificationsfordetailsaboutyourlamp(s).
c. Detection systems Inconventionalmicroscopy,fluorescenceisusuallydetectedwith
eitheraphotographic(35-mm)ordigitalcamera(whichfrequentlyharborsacooledcharge-coupleddevice,aCCD).Oneadvantageofa35-mmcameraisthatitcanrecordthe“true”color(asseenbyeyethroughthemicroscope)ofasinglefluorescentproteinaswellasthemixedcolorofcolocalizedfluorescentproteins.Butrecordinganimagewitha35-mmcameramayrequirealonger-than-averageexposuretime,effectivelyphotobleachingthesample.Digitalcameras,ontheotherhand,areusuallymoresensitivethan35-mmcamerasbut(unlessyouuseacolordigitalcamera)requireimageanalysissoftwaretoproduce“pseudo-colored”data.
d. Multicolor analysis Withthedevelopmentofoptimizedfiltersetsandtheintroduction
ofourredfluorescentprotein,DsRed,it’snowpossibletoseparateasmanyasthreefluorescentreporters(cyan,yellow,andred)bymicroscopy. Some investigators have even distinguished all fourcolors(cyan,green,yellow,andred)usingflowcytometry(Hawleyetal.,2001).Infact,manyexamplesofmulticoloranalysiscannowbefoundintheliterature(afewofwhicharelistedinTableIII),andwerecommendyousurveytheliteratureforhelpindecidingwhichcombinationwillworkbestforyou.Here,weofferthefollowingrec-ommendationsfortwo-coloranalysis:
• Fordual-labelingexperiments,werecommendusingDsRed2withEGFP.OneadvantageofusingDsRed2withEGFPisthatanoverlayoftheproteins’emissionsappearsyellow,soyoucandistinguishbetweentheindividualproteinsandtheoverlap.
• ECFPandEYFPcanalsobeusedtogethertocarryoutdouble-labelingexperiments(Greenetal.,2000;Ellenbergetal.,1999;July1999Clontechniques).WetestedtheopticalseparationofECFPandEYFPwithconventionalfluorescencemicroscopy.Inonestudy,weexaminedHeLacellsexpressingECFPandEYFP,targetedtothemitochondriaandnucleus,respectively(Greenetal.,2000).Tovisualizethefluorescence,weusedaZeissAxioskopequippedwitha100Wmercuryarclampandfilter sets for detecting ECFP (Omega Optical’s XF114) andEYFP(OmegaOptical’sXF104). Imageswerecapturedwitha35-mmcamera.Using thissetup, the twocolorsarewellseparated; no bleed-through was evident. During the initialexposure,(i.e.,duringthefirst~15sec),theseproteinsdisplay
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similarintensities;however,becauseECFPphotobleachesfaster than EYFP, unequal intensities become obvious afterseveralmoresecondsofillumination.
TABLE III. MULTI-COLOR AnALYSIS wITH LIVInG COLORS® PROTEInS
Type of Analysis Application Recommended References* Combination
Two-color Microscopy DsRed+EGFP(GFP) October1999Clontechniques Handler&Harrell,2001
ECFP+EYFP Greenetal.,2000 Stuurmanetal.,2000 October1999Clontechniques
Three-color Microscopy DsRed+ECFP+EYFP Bloembergetal.,2000 October1999Clontechniques
Four-color Flowcytometry DsRed+EGFP+EYFP Hawleyetal.,2001 +ECFP July2001Clontechniques
*Also,seePattersonetal.,(2001).
2. Laser-scanning microscopy Ifyouareusingalaser-scanningconfocalfluorescencemicroscope,
yourexcitationsourcewillusuallybeanargon-ionorkrypton-ionlaser,orboth.Withthe488-nmlineoftheargonlaser,youcanexciteEGFP,EYFP,andDsRed.Butyou’llneedthe458-nmlinefromanargonlaseror413-nmlinefromakryptonlasertoproducestrongemissionsfromECFP(Hawleyetal.,2001;Ellenbergetal.,1999).Thekrypton-ionlasermayalsobeusedtoexciteDsRed; its568-nmline isclosetoDsRed’s excitation maximum (Hawley et al., 2001). With so manylaser configurations now available, we recommend you consult themanufacturer(s)toobtainmoredetailedinformationaboutaparticularsetup.Comparethelaserspecificationswiththeexcitationspectraofthefluorescentproteinsyouplantouse.
3. Multi-photon laser-scanning microscopy Multi-photonexcitationmicroscopyhaswontheapprovalofmanyre-
searcherswhoseekotheralternativesforresolvingfluorescentlylabeledproteinsinvivo.Itsadvantageshavebeenwidelyreported(Piston,D.W.,1999;Potter,S.M.,1996;Marchant,J.S.,2001).Chiefamongthemis theability toexcitefluorophoreswith low-energy infrared light. IRlightnotonlypenetratestissuemoreeffectivelythanvisiblelight,italsominimizesphotobleachingandcauseslessphotodamage.Inprinciple,allLivingColorsFluorescentProteins,includingDsRed(Jakobsetal.,2000),aresuitableforuseinmulti-photonapplications.
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4. Fluorescence Microscopy Theprotocolprovidedbelowisonepossiblemicroscopyprocedure.
Otherequallysuitableandmoredetailedmicroscopyproceduresmaybefoundelsewhere(e.g.,Ausubeletal.,1995etseq.)
Materialsrequired: • 70%Ethanol • Dulbecco’sPhosphatebufferedsaline(DPBS;pH7.4) • DPBS/4%paraformaldehyde(pH7.4–7.6)
Add4gofparaformaldehydeto80mlofDPBS.Heattodissolve.Oncethesolutionhascooled,readjustthepHifnecessary,thendilutetoafinalvolumeof100ml.Storeat–20°C.
• Rubbercement,moltenagarose,orcommercialmountingmedium(e.g.,ProLong®AntifadeKit,MolecularProbes)
Fluorescencemicroscopyprocedure Inatissueculturehood: a.Sterilizeaglasscoverslipwith70%ethanol. b.Placethecoverslipinasteriletissueculturedish. c.Plateand transfect cells in the tissue-culturedish containing the
coverslip. note: Somecelltypesmaynotadheretotheglasscoverslip.Inthesecases,you
mayneedtopre-treattheglasscoverslipwithasubstratethatpromotescelladhesion(e.g.,lamin,orpoly-d-lysine,orboth).Asanalternativetoglasscoverslips,youmighttryculturingcellsdirectlyonBDFalconTMCultureSlides.
c.At theendofthecultureperiod,removethetissueculturemediaandwashoncewithDPBS.
d.Fixingcells i.AftercellshavebeenwashedwithDPBS,add freshlymade
DPBS/4%paraformaldehydedirectlytothecoverslip. ii.Incubatecellsinsolutionatroomtemperaturefor30min. iii.WashcellstwicewithDPBS.AllowcellstosoakinDPBSfor
10minduringeachwash. e.Mountingthecoverslipontoaglassmicroscopeslide i.Carefully remove the coverslip from the plate with forceps.
Paycloseattentiontowhichsideofthecoverslipcontainsthecells.
ii.Placeatinydropofcommercialmountingsolution(e.g., Pro-Long®AntifadeKit,MolecularProbes)ontheslide,andallowthecoversliptoslowlycontactthesolutionandtoliedownontheslide,cellsidedown.
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iii.Carefully aspirate the excess solution around the edge ofthecoverslipusingaPasteurpipetteconnectedtoavacuumpump.
iv.Ifdesired,sealthecoversliptothemicroscopeslideusingmoltenagarose,rubbercement,orblacknailpolish.
v.Allowtodry.Thedryingtimemayvarydependingonthemount-ingsolutionused.
vi.Examine slides by fluorescence microscopy. Once fixed,cellscanbestoredinthedarkat4°C.
B. Flow Cytometry 1. Multicolor analysis and cell sorting SeveralinvestigatorshavesortedGFP-expressingcellsbyflowcytometry
(Cheng&Kain,1995;Roppetal.,1995;Yu&vandenEngh,1995).FACSanalyseshavebeencarriedoutwithplantprotoplasts,yeast,E.coli,andmammaliancellsexpressingGFP(Atkins&Izant,1995;Feyetal.,1995;Sheenetal.,1995;Chengetal.,1996).MaximalemissionisachievedwhenEGFPisexcitedat488nm,whichcorrespondsperfectlytothe488-nmlineofargon-ionlasersusedinmanyflowcytometers.Infact,thisvariantwasselectedspecificallyonthebasisofitsincreasedfluorescenceinflowcytometryassays.
SinceEGFP,EYFP,andDsRedcanallbeexcitedbyasingle laserexcitationwavelength(488nm),two-color(EGFP/EYFP;Lybargeretal.,1998)andthree-color(Hawleyetal.,2001)flowcytometricanalysesarepossible.ECFPandEYFParenot recommended fordual-coloranalysiswithflowcytometrybecausetheargonlaser,usedforexcita-tioninmostflowcytometers,doesnotefficientlyexciteECFP.ToexciteECFP,yourflowcytometermustbeequippedwithakryptonlasertunedto413nm(Ellenbergeretal.,1998&1999)oranargonlasertunedto458nm(Hawleyetal.,2001).
EGFPandDsRedareperhapsthebestcombinationforcellsortingandtwo-coloranalysisbyflowcytometry.Theseproteinscanbeco-excitedbythe488nmlineoftheargonlaserandtheiremissionscanbecon-venientlyrecordedthroughtheFL-1(green)andFL-2(red)channels.Theanalysiscanbefurtherrefinedbyusingoptimizedfilterssetsandbyadjustingthecompensationsettings.
IV. Detection of GFP, GFP Variants, and DsRed continued
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IV. Detection of GFP, GFP Variants, and DsRed continued
2. Detecting fluorescent proteins in ethanol-treated cells Whenexpressedasaqueoussolubleproducts,fluorescentproteins
mayleakfromethanol-treatedcells;ethanolpermeabilizestheplasmamembrane.Toavoidthisproblem,wesuggestyouuseparaformalde-hydeifyouplantofixfluorescentprotein-expressingcells.Otherwise,ifyouwishtomeasuretransfectionefficiency,butplantofixcellswithethanol,werecommendusingEGFP-F,ECFP-Mem,orEYFP-Mem,describedbelow.
Insomecases,youmaynoticeagradualincreaseinbackgroundfluo-rescence inethanol-treatedcellsduringflowcytometry.ArinsewithPBSsupplementedwith1%BSAafterethanolfixation,orlongfixationtimes(18–24hr),eliminatesthisproblem(D.Ucker,pers.comm.).
a.EGFP-Fispost-translationallyfarnesylated,soitremainsattachedtotheinnerfaceoftheplasmamembraneevenduringtreatmentwithethanol.EGFP-Fcontainsashort,C-terminalfarnesylationsignalfromc-Ha-Ras(Aronheimetal.,1994;Hancocketal.1991).ThesignaltargetsEGFP-FtotheplasmamembranewithoutalteringtheintrinsicfluorescentpropertiesoftheEGFPchromophore(Jiang&Hunter,1998).
b.ECFP-Mem&EYFP-MemareN-terminalfusionsthatcontaintheN-terminal20aminoacidsofneuromodulin(GAP-43;Moriyoshietal.,1996).Duringpost-translationalprocessing,apalmitoylgroupisaddedtotheneuromodulindomainofthefusionprotein.Themodi-ficationtargetsthefluorescentproteinprimarilytotheinnerfaceoftheplasmamembrane.Asmallamountofthefusiondoesbindothercellularmembranes.
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IV. Detection of GFP, GFP Variants, and DsRed continued
C. Quantitative Fluorometric AssayRecombinantGFP(orEGFP)canbeusedinfluorometricassaystoconfirmandmeasureGFPexpression.Aftergeneratingastandardcurveusingknownamountsofrecombinantprotein,comparethefluorescenceintensitiesofyourexperimentalsamplestothestandardcurvetodeterminetheamountofGFPexpressedinthesample.AdetailedmethodforquantitationofwtGFPlevelsusingtheTD-700fluorometerisavailablefromTurnerDesigns,Inc(www.turnerdesigns.com).
Whengeneratingthestandardcurve,therecombinantproteinshouldbedilutedinthesamebufferastheexperimentalsamples.Insomeexpressionsystems,GFPcanbedetecteddirectlyinthefluorometer(simplysuspendcellsinanisotonicbuffersuchasPBS);inothercases,cellswillneedtobedisruptedinordertooptimallydetectGFPfluorescence.
Figure6showsacomparisonofthefluorescenceintensityofGFPdetectedinsonicationbufferand inaclearedbacterial cell lysate.These resultsillustratethatthesensitivityandthelinearrangeofdetectionofGFParerelativelyunaffectedbythecelllysatebackground.
Figure 6. Comparison of fluorescence intensity of GFP in sonication buffer versus GFP in cell lysates.LBwasinoculatedwithacultureofuntransformedJM109cellsandincubatedovernightat30°C.Celllysateswerepreparedasdescribedintheprotocol.SerialdilutionsofrGFPwerepreparedinbothsonicationbufferandJM109celllysate.FluorescenceintensitywasmeasuredinamodifiedHoeferPharmaciaBiotech,Inc.DyNAQuant200Fluorometerusinganexcitationfilterof365nmandanemissionfilterof510nm.(RFU=relativefluorescenceunits)
100
1
2
�
4
2
Log GFP (ng)
GFP in 6.2� mg JM109 lysate
GFP in sonication buffer
Log
RFU
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ThefollowingprocedureshavebeendevelopedusingourRecombinantGFPProtein(rGFP#8360-2).Useanexcitationfilterof365nmandanemissionfilterof510nmwhenassayingwtGFPorGFPuv,andanexcitationfilterof450–490nmandanemissionfilterof510nmwhenassayingEGFP.
1.Materialsrequired • Sonicationbuffer(pH8.0) 50 mM NaH2PO4 10 mM Tris-HCl(pH8.0) 200 mM NaCl AdjustpHto8.0.Storeatroomtemperature. • PBS(pH7.4) 2.GeneratingaGFPstandardcurve: a.PrepareserialdilutionsofrGFPinsonicationbuffer,deionizedH2O,
or10mMTris-HCl(pH8.0). note: Samplescanbestoredat–20°C. b.Assaydilutionsinafluorometer. c.Plotthelogoftherelativefluorescenceunits(RFU)asafunctionof
thelogoffold-dilutionofeachsamplesuchasthestandardcurveshowninFigure7.
3.Measuringfluorescenceintensityofbacterialsamples: a.Pellet20mlofbacterialcellculture. b.Removethesupernatantandfreezepelletat–70°C. c.Washpelletoncewith1.6mlofPBS. d.Resuspendpelletin1.6mlofsonicationbuffer. e.Prepare2-foldserialdilutionsinsonicationbuffer. f.Assaydilutionsinafluorometer. g.Compareresultswiththoseofthestandardcurvetodeterminethe
amountofGFPproteinexpressedintheexperimentalsamples. 4.Preparationofbacterialcelllysates: a.Pellet100mlofbacterialcellcultureandremovethesupernatant. note:Cellpelletsmaybestoredat–70°Cforlateruse. b.Washpellettwicewith4mlofPBS. c.Resuspendpelletin4mlofsonicationbuffer. d.Freeze/thawsampleinadryice/ethanolbathfivetimes. e.Vortexsamplefor1min,thenincubateonicefor1min. f.RepeatStepetwomoretimes. g.Runsamplethroughan18-gaugeneedleseveraltimes.
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Figure 7. Relative fluorescence intensity of two-fold serial dilutions of a suspension of GFP-transformed cells in sonication buffer. LBwasinoculatedwithJM109cellstransformedwithaplasmidencodingwtGFPandincubatedovernightat30°C.Fluorescenceintensitywasmeasuredbyreading5-µlsamplesof2-folddilutionsinamodifiedHoeferPharmaciaBiotech,Inc.DyNAQuant200Fluorometerusinganexcitationfilterof365nmandanemissionfilterof510nm.
00
1
2
1
Log Fold Dilution
DR60�14pr.bw/GFP.fi.sb
Log
RFU
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h.Transfercelllysatetoa1.5-mlmicrocentrifugetubeandcentrifugeat12,000rpmfor5minat4°C.
i.Collectthesupernatantanddeterminetheproteinconcentrationus-ingastandardassay(e.g.,Bradford,Lowry,etc.;Scopes,1987).
j.Assaythedilutionsinafluorometer. k.Comparetheresultswiththestandardcurvetodeterminetheamount
ofGFPexpressedintheexperimentalsamples.
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A. General InformationOurRecombinantGreenFluorescentProtein(rGFP;#8360-2),RecombinantEGFP(rEGFP;#8365-1),andRecombinantGFPuv(rGFPuv;#8366-1)arepurifiedfromtransformedE.coliusingamethodwhichensuresoptimalpurityoftherecombinantproteinandmaintenanceofGFPfluorescence.Allproteinsare27-kDamonomerswith239aminoacids.TheexcitationandfluorescenceemissionspectrafortherGFPisidenticaltoGFPpurifiedfromAequoreavictoria(Chalfieetal.,1994).Thepurifiedproteinsretaintheirfluorescencecapabilityundermanyharshconditions (Section II.F)andaresuitableascontrolreagentsforGFPexpressionstudiesusingtheLivingColorslineofGFPreportervectors.Applications of purified rGFP and rGFP variant proteins include useas standards for SDS or two-dimensional PAGE, isoelectric focusing,Westernblotanalysis,calibrationoffluorometersandFACSmachines,fluo-rescencemicroscopy,andmicroinjectionofGFPintocellsandtissues.
B. rGFP and rGFP Variants as Standards in western BlotsWhenusedasastandardforWesternblottingapplicationsinconjunctionwiththeLivingColorsA.v.PeptideAntibody(#8367-1,-2),therecombinantproteinscanbeusedtocorrelateGFPexpressionlevelstofluorescenceintensityortodifferentiateproblemswithdetectionofGFPfluorescencefromexpressionofGFPprotein.Useastandardprocedurefordiscontinuouspolyacrylamidegelelectropho-resis(PAGE)toresolvetheproteinsonaone-dimensionalgel(Laemmli,1970).Ifanotherelectrophoresissystemisemployed,thesampleprepara-tionshouldbemodifiedaccordingly.Justpriortouse,allowrGFPproteintothawatroomtemperature,mixgentlyuntilsolution isclear,andthenplacetubeonice.Onaminigelapparatus,25–75µgof lysateproteinper lane is typicallyneededforsatisfactoryseparation(i.e.,discretebandingthroughout themolecularweightrange)ofaproteinmixturederivedfromawholecellortissuehomogenate.IfrGFPistobeusedasaninternalstandardinaCoo-massieblue-stainedminigel,werecommendloading500ngofrGFPperlane.IfrGFPisaddedtoatotalcell/tissuelysateorothercrudesample,theamountoftotalproteinloadedperlanemustbeoptimizedfortheparticularapplication.ForWesternblottingapplications,werecommendloading40–400pgofrGFP(orrGFPvariant)perlaneforastrongpositivesignal.Generally,rGFPwillnotfluoresceonanSDSgeloraWesternblot.
V. Purified Recombinant GFP and GFP Variants
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C. rGFP and rGFP Variants as a Control for Fluorescence MicroscopyThefollowingtwoprotocolsareforuseofrGFPorrGFPvariantsasacon-trolonmicroscopeslidesinfluorescencemicroscopy.ThepurifiedproteinsmaybeusedtooptimizelampandfiltersetconditionsfordetectionofGFPfluorescence,orasaqualitativemeanstocorrelateGFPfluorescencewiththeamountofproteinintransfectedcells.
1. Unfixed samples Usethismethodforlivecellfluorescenceorothercaseswhereafixa-
tionstepisnotdesired. a.Perform1:10serialdilutionsofthe1.0mg/mlrGFPorrGFPvariant
stocksolutionwith10mMTris-HCl(pH8.0)toyieldconcentrationsof0.1mg/mland0.01mg/ml.
notes: • Thesedilutionsshouldsufficeasapositivecontrol.The1.0mg/mlsolutionwill
giveaverybrightfluorescentsignalbymicroscopy. • Thediluted samples canbealiquotedandstored frozenat –70°C for up to
1yrwithnolossoffluorescenceintensity.Avoidrepeatedfreeze-thawcycleswiththesamealiquot.
b.Using a micropipette, spot 1–2 µl of diluted protein onto themicroscopeslide.Ifusingaslidethatcontainsamountedcoverslip,positionthespotseveralmillimetersawayfromthesamplesuchthatasecondcoverslipcanbeaddedovertheproteinspot.
c.Allow the protein to air-dry for a few seconds, and mark thepositionofthespotontheothersideoftheslidetoaidinfocusing.
d.Add a coverslip over the spot using a 90% glycerol solution in100mMTris-HCl(pH7.5).
e.Fluorescencefromthespotisbestviewedatlowmagnification,usingeithera10Xor20Xobjectivelens.
2. Fixed samples Insomecasesitmaybenecessarytofixtherecombinantproteinto
themicroscopeslidepriortomicroscopy.Thiscanbedonebydippingthesectionofthemicroscopeslidecontainingtheair-driedproteinspot(afterStep1.cabove)into100%methanolfor1min.AllowtheslidetodrycompletelyandplaceacoverslipoverthesampleasinStep1.dabove.
V. Purified Recombinant GFP and GFP Variants continued
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A. Applications for GFP Antibodies WeofferthreeGFP-specificantibodies:
• Living Colors®A.v. PeptideAntibody (#8367-1, -2) detects all GFPvariants.TheA.v.PeptideAntibody,whichisalsoavailableconjugatedtohorseradishperoxidase(HRP),isrecommendedforWesternanaly-sis.
• LivingColors®A.v.(JL-8)MonoclonalAntibody(#8371-1,-2),amousemonoclonal(subclassIgG2a),recognizesallLivingColorsGFPvari-ants,includingourenhancedanddestabilizedproteins,fusionstotheseproteins,andpurifiedrecombinantGFP.TheJL-8MonoclonalisidealforWesternandELISAapplications.
• LivingColors®Full-LengthA.v.PolyclonalAntibody(#8372-1,-2)wasspecifically developed and tested for immunoprecipitating GFP andGFPvariants. ItalsodetectsdestabilizedvariantsandfusionstoallLivingColorsGFPproteins.
WealsoofferDsRed-specificantibodies: • Living Colors® D.s. Peptide Antibody (#8370-1, -2) recognizes wt
DsRedandDsRedvariants suchasDsRed1-E5andDsRed2.TheantibodyalsobindsDsRedfusionswhentheproteinofinterestisfusedtotheN-terminusofDsRedoroneof itsvariants.BecausetheD.s.PeptideepitopeoftenbecomeshiddenwhenproteinsarefusedtotheC-terminus of DsRed, you may have difficulty detecting C-terminalfusionswiththisantibody.Raisedinrabbitsandaffinity-purified,thispolyclonalantibodyisrecommendedforuseinWesternblottingandimmunoprecipitation.TheD.s.PeptideAntibodydoesnotcross-reactwithanyAequoreavictoriaGFPvariants.
• LivingColors®DsRedMonoclonalAntibody(#8374-1,-2)recognizesdenaturedformsofwild-typeDsRedanditsvariants,includingDsRed1,DsRed2,andDsRed1-E5.RecommendedforWesternblottingapplica-tionsonly,thisantibody(mouseIgG)bindsDsRed1andDsRed2evenwhen theproteinsareexpressedas fusions tootherproteins.BothN-andC-terminalfusionsarerecognized.
VI. GFP Antibodies
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VI. GFP Antibodies continued
B. Immunoprecipitation with the Living Colors A.v. Peptide Antibody 1. Solutions Required • PBS • Lysisbuffer PBScontaining0.5%TritonX-100,5mMEDTA,andthefollow-
ingproteaseinhibitors(allavailablefromSigma):PMSF,0.1mM;pepstatinA,10µM;leupeptin,10µM;aprotinin,25µg/ml.
• LivingColorsPeptideAntibody • ProteinAorProteinGagarosebeads(PierceorBioRad) Use20–30µlofbeadspersample.Spindownbeadstoremove
theethanol,thenwashbeadstwiceinPBS.Aspiratethesuperna-tantfromthepelletofbeads.
2. Procedure a.Spindown~1x106cells(orcellsfromone100-mmdish)andwash
twiceinPBS.Resuspendin1.2mloflysisbuffer. b. Incubateat4°Cfor30minwithcontinuousgentleinversion. c. Clearthelysatebycentrifuginginamicrocentrifugeat12,000rpm
for20minat4°C. d. Transfer1mlofsupernatanttoa1.5-mlmicrocentrifugetube. e. Add5µgofLivingColorsA.v.PeptideAntibodytothesupernatant.
Incubateat4°Cfor2–3hrwithcontinuousgentleinversion. f. Addtheantibody/lysatemixturetothepelletofProteinA(orProtein
G)agarosebeads. g. Vortexandincubateat4°Cfor2hrwithcontinuousgentleinver-
sion. h. Washthebeads5timeswithPBS.Foreachwash,add1mlofPBS
andincubatefor2minwithcontinuousgentleinversion.Aspiratethesupernatantfromthepelletofbeads.
3. Sample analysis: a. Washthebeadswith1mlofLysisBuffer. b. Repeatthiswashprocedurefourtimes. c. Add30µlof2XSDS-PAGEsamplebuffertobeads. d. Vortex,thenboilfor2min. e. Repeatstepd. f. Centrifuge inamicrocentrifugeat12,000rpmfor1minat room
temperature. g. Electrophorese10–20µlofsupernatantonanSDS-polyacrylamide
gel(a12%gelwillresolveGFPat27kDa).AnalyzetheresolvedproteinsonaWesternblot,bysilverorCoomassiebluestaining,orbyautoradiography.
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VII. Troubleshooting Guide
A. Potential Difficulties in Using GFP Fluorescence • VariabilityintheintensityofGFPfluorescencehasbeennoted.Thismay
bedueinparttotherelativelyslowformationoftheGFPchromophoreandtherequirementformolecularoxygen(Heimetal.,1994).
• The slow rate of chromophore formation and the apparent stabilityofwtGFPmayprecludetheuseofGFPasareportertomonitorfastchangesinpromoteractivity(Heimetal.,1994,Davis,I.etal.,1995).ThislimitationisreducedbyuseofEGFPanddestabilizedGFPs,whichacquirefluorescencefasterthanwtGFP(Heimetal.,1994).
• ThewtGFPcodingsequencescontainacrypticplantintron(betweennucleotides400and483)thatisefficientlysplicedoutinArabidopsisandresultsinanonfunctionalprotein(Haseloff&Amos,1995).FunctionalwtGFPhasbeentransientlyexpressedinseveralotherplantspeciesindicatingthatthecrypticintronmaynotberecognizedorrecognizedlessefficientlyinthesespecies.EGFP,ECFP,andEYFPdonotcontainthecrypticintronandcanbeusedforexpressioninplants.
• SomepeoplehaveputGFPexpressionconstructsintotheirsystemandfailedtodetectfluorescence.Therecanbenumerousreasonsforfailure,includinguseofaninappropriatefilterset,expressionofGFPbelowthelimitofdetection,andfailureofGFPtoformthechromophore.RecombinantGFPProteinandaGFP-specificantibody,suchastheLivingColorsA.v.PeptideAntibody,maybeusedtotroubleshootGFPexpressioninthesecases.SinceGFPuvandEGFPfluorescebrighterthanwtGFP,theyarebetteralternativesforexpressionofGFP.
• GFPtargetedtoalowpHenvironmentmaylosefluorescence. • Ethanol-fixed cells will lose fluorescence because the soluble fluo-
rescentproteinleaksthroughthepermeabilizedplasmamembranes.Use pEGFP-F, pECFP-Mem, or pEYFP-Mem Vectors to avoid thisproblem.
B. Requirements for GFP Chromophore Formation • FormationofthechromophoreinwtGFPappearstobetemperature
sensitive;however,themutationsinEGFP,EYFP,ECFP,andGFPuvsuppressthisthermosensitivity.Insomecases,E.coli,yeast,andmam-maliancellsexpressingwtGFPhaveshownstrongerfluorescencewhengrownatlowertemperatures(Heimetal.,1994;Ward,pers.comm.;Limetal.,1995;Pines,1995;Ogawaetal.,1995).Hence,incubationatalowertemperaturemayincreasethefluorescencesignalobtainedwhenusingwtGFP.
• GFPchromophoreformationrequiresmolecularoxygen(Heimetal.,1994;Davis,D.F.etal.,1995);therefore,cellsmustbegrownunderaerobicconditions.
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VII. Troubleshooting Guide continued
C. Photobleaching or Photodestruction of Chromophore • Exciteat470nmforwtGFP,360–400nmforGFPuv,and488nmfor
thered-shiftedGFPexcitationvariants.Excitationatthe395-nmpeakforwtGFPmayresultinrapidlossofsignal.ForGFPuv,usethelongestpossibleexcitationwavelength tominimize the rateofphotobleach-ing.
•Atungsten-QTHorargonlightsourceispreferable.MercuryandxenonlampsproducesignificantUVradiationwhichwillrapidlydestroythechromophoreunlessstronglyblockedbyappropriatefilters.
D. Autofluorescence • Somesamplesmayhaveasignificantbackgroundautofluorescence,
e.g.,wormguts(Chalfieetal.,1994;Niswenderetal.,1995).Aband-passemissionfiltermaymaketheautofluorescenceappearthesamecolorasGFP;usingalong-passemissionfiltermayallowthecoloroftheGFPandautofluorescencetobedistinguished.UseofDAPIfiltersmay also allow autofluorescence to be distinguished (Brand, 1995;Pines,1995).
• Mostautofluorescenceinmammaliancellsisduetoflavincoenzymes(FAD and FMN;Aubin, 1979) which have absorption and emissionmaximaat450and515nmrespectively.ThesevaluesareverysimilartothoseforwtGFPandthered-shiftedGFPvariants,soautofluores-cencemayobscuretheGFPsignal.TheuseofDAPIfilterswhenusing450/515nmlightforexcitationmaymakeautofluorescenceappearbluewhiletheGFPsignalremainsgreen.Inaddition,somegrowthmediacancauseautofluorescence.Whenpossible,performmicroscopyinaclearbuffersuchasPBS,ormediumlackingphenolred.Also,whenselecting forGFPexpression inmammaliancells,youmaywant toexcludethehighestexpressingclones,whichwillexhibitmoreback-groundfluorescenceinculture(D.Piston,pers.comm.).
• Some cell types can produce a speckled autofluorescence patternwhichislikelyduetomitochondriallyboundNADH(Aubin,1979).Thisproblemisminimizedwithexcitatorylightaround488nm,asopposedtoUVexcitation(Niswenderetal.,1995).Therefore,werecommendusingEGFPforexpressioninthesesystems.
• Formammaliancells,autofluorescencecanincreasewithtimeincul-ture.Forexample,whenCHOorSCIcellswereremovedfromfrozenstocksandreintroducedintoculture,theobservedautofluorescence(emissionat520nm)increasedwithtimeuntilaplateauwasreachedaround48hours(Aubin,1979).Therefore,insomecasesitmaybepreferabletoworkwithfreshlyplatedcells.
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VII. Troubleshooting Guide continued
• Alwaysuseamock-transfectedcontroltogaugetheextentofautofluo-rescence.
• For fixed cells, autofluorescence can be reduced by washing with0.1%sodiumborohydrideinPBSfor30minafterfixation.
E. Considerations for Mammalian Expression • Useanenhancedfluorescentvariant(ECFP,EGFP,orEYFP)instead
ofwtGFP.Thebrighterfluorescenceandimprovedtranslationofthesevariantsmakesthemeasiertodetect.
•VerifyyourGFPvariantplasmidconstructandconcentrationwitharestrictiondigest.Verifythatallsubcloningstepshavebeendonecor-rectly,keepinginmindspecificrestrictionsitesinsomevectorswhichareinactivatedbymethylation.OurEGFPSequencingPrimersmaybeusedtoverifysequencejunctions.
• Besurethevectoriscompatiblewithyourcelltype.FindoutiftheCMVpromoterhasbeenusedpreviously for transientexpression inyourcells.
•Useanotherassaytoestimatetransfectionefficiency.Transfectwithasecondreporterplasmidthatcontainsthesamepromoterelement.Forexample,usepEGFPLuc(#6169-1)orpHygEGFP(#6014-1).WithpEGFPLuc (#6169-1), you can determine transfection efficiency byfluorescencemicroscopyandobtainquantitativedatausingastandardluciferaseassay.
•ExpressionofGFPproteincanbeverifiedbyWesternanalysisusingeithertheLivingColorA.v.PeptideAntibody(#8367-1,-2)ortheLivingColorsFull-LengthA.v.PolyclonalAntibody(#8373-1,-2).
F. Optimizing Microscope/FACS Applications • Ingeneral,optimalvisualdetectionofGFPfluorescencebymicroscopy
isachievedinadarkenedroom,afteryoureyeshaveadjusted. •ChoosethefiltersetthatisoptimalfortheGFPvariantthatyouareus-
ing.Ingeneral,conditionsusedforfluoresceinshouldgivesomesignalwithallvariantsexceptGFPuv.Autofluorescencemaybeaprobleminsomecelltypesororganisms.FormoreinformationonthefiltersetsrecommendedforGFPanditsvariants,seeSectionIV.
•Tocheckthemicroscopesetup,spotasmallvolumeofpurifiedrecom-binantGFPprotein(e.g.,rGFP,rEGFP,orrGFPuv)onamicroscopeslide.Asacrudesubstitute,anysourceoffluoresceincanbeused,suchasafluorescein-conjugatedantibodyoravidin-fluorescein.
• GFPfluorescenceisverysensitivetosomenailpolishesusedtosealcoverslips(Chalfieetal.,1994;Wang&Hazelrigg,1994).Inplaceofnailpolishformountingcoverslips,werecommendmoltenagarose,rubbercement,orcommercialmountingsolution.
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• ExcitingGFPintenselyforextendedperiodsmaygeneratefreeradicalsthataretoxictothecell.Thisproblemcanbeminimizedbyexcitationat450–490nm.
G. Anomalous Band in some GFP Plasmid Preparations Wehaveoccasionallyobservedabandrunningatapproximately500bp
insome(butnotall)plasmidpreparationsoftheGFPvectorsthathavethebackbonecarryingthekanamycinresistancegeneandthef1origin(i.e.,pEGFP-1,pEGFP-N1,-N2,-N3,-C1,-C2,-C3,pEYFP-C1,-N1,pEYFP-1,andpECFP-N1,-C1,and-1).ThepresenceandlevelofthisbandvariesgreatlyfromoneplasmidDNApreparationtoanother.Wehavenotthoroughlycharacterizedthisband;however,itappearstobeasmallcircularDNA,possiblysingle-stranded,generatedasabyproductofplasmidreplication.Asfaraswecanascertain,thisbanddoesnotinterferewithligatinginsertsintothevectors,doesnotaffecttransformationortransfectionefficiencies,anddoesnothaveanydeleteriouseffectonmammaliancells.However,ifyouwishtoavoidthepresenceofthisbandinyourplasmidpreps,tryanynon-alkalinebacteriallysisprocedure(Sayersetal.,1996)orgel-purifythemajorbandafteralkalinelysis.
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VIII. References
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Heinlein,M.,Epel,B.L.,Padgett,H.S.&Beachy,R.N.(1995)Interactionoftobamovirusmovementproteinswiththeplantcytoskeleton.Science270:1983–1985.Htun,H.,Barsony,J.,Renyi,I.,Gould,D.&Hager,G.L.(1996)Visualizationofglucocorticoidreceptortranslocationandintranuclearorganizationinlivingcellswithagreenfluorescentproteinchimera.Proc.Natl.Acad.Sci.USA93:4845–4850.Inouye,S.&Tsuji,F.I.(1994a)Aequoreagreenfluorescentprotein:Expressionofthegeneandfluorescentcharacteristicsoftherecombinantprotein.FEBSLetters341:277–280.Inouye,S.&Tsuji,F.I.(1994b)EvidenceforredoxformsoftheAequoreagreenfluorescentprotein.FEBSLetters351:211–214.Jacobi,C.A.,Roggenkamp,A.,Rakin,A.&Heesemann,J.(1995)YersiniaenterocoliticaYopE-GFPhybridproteinexpressioninvitroandinvivo.PosterpresentationatFluorescentProteinsandApplicationsMeeting,PaloAlto,CA.Jakobs S., Subramaniam, V., Schonle,A., Jovin,T. M. & Hell, S. W. (2000) EFGP and DsRedexpressingculturesofEscherichiacoliimagedbyconfocal,two-photonandfluorescencelifetimemicroscopy.FEBSLett.479:131–135.Jiang,W.&Hunter,T.(1998)Analysisofcell-cycleprofilesintransfectedcellsusingamembrane-targetedGFP.BioTechniques24:349–350.Kaether,C.&Gerdes,H.-H.(1995)Visualizationofproteintransportalongthesecretorypathwayusinggreenfluorescentprotein.FEBSLetters369:267–271.Kahana,J.A.,Schnapp,B.J.&Silver,P.A.(1995)Kineticsofspindlepolebodyseparationinbud-dingyeast.Proc.Natl.Acad.Sci.USA92(21):9707–9711.Kain,S.R.,Mai,K.&Sinai,P.(1994)Humanmultipletissuewesternblots:anewimmunologicaltoolfortheanalysisoftissue-specificproteinexpression.BioTechniques17:982–987.Kain,S.R.,Adams,M.,Kondepudi,A.,Yang,T.T.,Ward,W.W.&Kitts,P.(1995)Thegreenfluorescentproteinasareporterofgeneexpressionandproteinlocalization.BioTechniques19:650–655.Kerrebrock,A. W., Moore, D. P., Wu, J. S. & Orr-Weaver,T. L. (1995) Mei-S332, a Drosophilaproteinrequiredforsister-chromatidcohesion,canlocalizetomeioticcentromereregions.Cell83:247–256.Kneen,M.,Farinas,J.,Li,Y.&Verkman,A.S.(1998)GreenfluorescentproteinasanoninvasiveintracellularpHindicator.Biophys.J.74:1591–1599.Kozak,M.(1987)Ananalysisof5’-noncodingsequencesfrom699vertebratemessengerRNAs.NucleicAcidsRes.15:8125–8148.Laemmli,U.K.(1970)Cleavageofstructuralproteinsduringtheassemblyoftheheadofbacterio-phageT4.Nature227:680–685.Lewis,P.J.&Errington,J.(1996)Useofgreenfluorescentproteinfordetectionofcell-specificgeneexpressionandsubcellularproteinlocalizationduringsporulationinBacillussubtilis.Microbiology142:733–740.Li,X.,Zhang,G.,Ngo,N.,Zhao,X.,Kain,S.R.&Huang,C.-C.(1997)DeletionsoftheAequoreavictoriagreenfluorescentproteindefinetheminimaldomainrequiredforfluorescence.J.Biol.Chem.272:28545–28549.Li, X., Zhao, X., Fang,Y., Jiang, X., Duong,T., Huang, C.-C. & Kain, S. R. (1998) Generationof destabilized enhanced green fluorescent protein as a transcription reporter. J. Biol. Chem.273:34970–34975.Lim,C.R.,Kimata,Y.,Oka,M.,Nomaguchi,K.&Kohno,K.(1995)Thermosensitivityofgreenfluores-centproteinfluorescenceutilizedtorevealnovelnuclear-likecompartmentsinamutantnucleoporinNsp1.J.Biochem.118:13–17.LivingColorsDestabilizedECFP&EYFPVectors.(July1999)ClontechniquesXIV(3):14–15.LivingColorsRedFluorescentProtein.(October1999)ClontechniquesXIV(4):2–6.LivingColorsDestabilizedEGFPVectors.(April1998)ClontechniquesXIII(2):16–17.
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IX. Related Products
ForthelatestandmostcompletelistingofallClontechproducts,pleasevisitwww.clontech.com
WeofferavarietyofvectorsforexpressionofGFPanditsvariants.TheVectorInformationPacket thataccompaniesanypurchasedvectorprovidesamap,multiplecloningsite(MCS),completesequence,andadditionalinformationaboutthevector.Muchofthisinformationisalsoavailableatourwebsite.
Bacterial Expression VectorsProteincanbeoverexpressedinbacteriausingthesevectors.Thecodingse-quencecanbeexcisedusingrestrictionsitesintheflankingMCSregions;orthecodingsequencecanbeamplifiedbyPCR.• pEGFPVector 6077-1• pEYFPVector 6004-1• pECFPVector 6075-1• pGFPVector 6097-1• pGFPuvVector 6079-1
Mammalian Expression Vector• pCMS-EGFP 6101-1
n-Terminal Protein Fusion Vectors• pECFP-N1 6900-1• pEYFP-N1 6006-1• pEGFP-N1/2/3 * 6085-1,6081-1,6080-1
C-Terminal Protein Fusion Vectors• pECFP-C1 6076-1• pEYFP-C1 6005-1• pEGFP-C1/2/3 * 6084-1,6083-1,6082-1 *EachvectorhastheMCSinoneofthreepossiblereadingframes
Promoterless Mammalian Expression VectorsUsedformonitoringtheactivityofpromotersorpromoter/enhancercombinationsinsertedintotheMCSupstreamofthegeneencodingtheGFPvariant.• pECF