Living Colors User Manual Volume II - Molecular · PDF fileto the Indian and Pacificoceans...
Transcript of 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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Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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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Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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.
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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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Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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
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II. Properties of Reef Coral Fluorescent Proteins continued
TAB
LE
II. P
HO
TOP
Hy
SIC
AL
PR
OP
ER
TIE
S O
F R
EE
F C
OR
AL
FL
uO
RE
SC
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TE
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Pro
tein
E
xcit
atio
n
Em
issi
on
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ime
to d
etec
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Bri
gh
tnes
s Q
uat
ern
ary
uti
lity
as a
u
tilit
y in
max
(n
m)
max
(n
m)
(hr)
re
lati
ve t
o E
GF
P
stru
ctu
re
rep
ort
er
fusi
on
s
Ree
f C
ora
l Flu
ore
scen
t P
rote
ins
Am
Cya
n1a
458
489
8–12
+
++
Te
tram
er
++
+
+
ZsG
reen
1a 49
350
58–
12
++
++
Te
tram
er
++
++
+
ZsY
ello
w1a
529
539
8–12
+
+
Tetr
amer
+
++
+
DsR
ed-E
xpre
ss
557
579
8–12
+
++
Te
tram
er
++
+
++
DsR
ed2
563
582
24
++
+
Tetr
amer
+
++
+
+
AsR
ed2a
576
592
8–12
+
+
Tetr
amer
+
++
+
HcR
ed1
588
618
16
+
Dim
er
+
++
+
Aeq
uo
rea
Vic
tori
a G
FP
var
ian
ts
EC
FP
43
947
68–
12
+
Mon
omer
+
+
++
+
EG
FP
48
451
08–
12
++
+
Mon
omer
+
++
+
++
+
EY
FP
51
252
98–
12
++
+
Mon
omer
+
++
+
++
+a
Pro
neto
agg
rega
tion
b A
sm
easu
red
byF
AC
Sa
naly
sis
usin
gtr
ansi
ently
tran
sfec
ted
mam
mal
ian
cell
cultu
res
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Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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.
II. Properties of Reef Coral Fluorescent Proteins continued
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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
II. Properties of Reef Coral Fluorescent Proteins continued
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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.
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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.
II. Properties of Reef Coral Fluorescent Proteins continued
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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).
II. Properties of Reef Coral Fluorescent Proteins continued
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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.
II. Properties of Reef Coral Fluorescent Proteins continued
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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
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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.
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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
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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
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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
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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.
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Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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.
IV. Detection of Reef Coral Fluorescent Proteins continued
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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).
IV. Detection of Reef Coral Fluorescent Proteins continued
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IV. Detection of Reef Coral Fluorescent Proteins continued
TAB
LE
VIII
. LIV
ING
CO
LO
RS
® A
NT
IBO
DIE
S
An
tib
od
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An
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S
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t
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(JL-
8)
Affi
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sem
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yclo
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382
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ll-le
ngth
GF
Pp
rote
in
A.v
.Pep
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ibod
y(p
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6
3237
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finity
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tide
Ab'
sto
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nize
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and
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632
397
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sva
riant
s
HcR
edP
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lAnt
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6324
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d
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 �� Version No. PR37085
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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
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Living Colors® User Manual Vol. II: 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
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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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Aronheim,A.,Engelberg,D.,Li,N.,al-Alawi,N.,Schlessinger,A.&Karin,M. (1994)MembranetargetingofthenucleotideexcahngefactorSosissufficientforactivatingtheRassignalingpathway.Cell78:949–961.
Atkins,D.&Izant,J.G.(1995)ExpressionandanalysisofthegreenfluorescentproteingeneinthefissionyeastSchizosaccharomyces pombe. Curr. Genet. 28:585–588.
Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G.,Smith,J.A.&Struhl,K.(1994)InCurrent Protocols in Molecular Biology(JohnWileyandSons,NY),Vol.1,Ch.5and9.
Bevis,B.J.,Glick,B.S.(2002)RapidlymaturingvariantsoftheDiscosomaredfluorescentprotein(DsRed).Nat Biotechnol20:83–7.
Baird,G.S.,Zacharias,D.A.&Tsien,R.Y.(2000)Biochemistry,mutagenesis,andoligomerizationofDsRed,aredfluorescentproteinfromcoral.Proc. Natl. Acad. Sci. USA97:11984–11989.
Carey,K.L.,Richards,S.A.,Lounsbury,K.M.&Macara, I.G. (1996)Evidenceusingagreenfluorescentprotein-glucocorticoidreceptorthattheRAN/TC4GTPasemediatesanessentialfunctionindependentofnuclearproteinimport.J. Cell Biol. 133(5):985–996.
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VII. References
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 �� Version No. PR37085
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Gurskaya,N.G.,Fradkov,A.F.,Terskikh,A.,Matz,M.V.,Labas,Y.A.,Martynov,V.I.,Yanushevich,Y.G.,Lukyanov,K.A.&Lukyanov,S.A.(2001)GFP-likechromoproteinsasasourceof far-redfluorescentproteins.FEBS Lett.507:16–20.
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VII. References continued
Protocol No. PT3404-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR37085 �7
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 �8 Version No. PR37085
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
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VIII. Related Products
Protocol No. PT3404-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR37085 ��
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
Notes
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3404-1 30 Version No. PR37085
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
Notes
Protocol No. PT3404-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR37085 31
Living Colors® User Manual Vol. II: Reef Coral Fluorescent Proteins
Notes