Type:RabbitIgG
Applications:IF;WB
E=ELISA;FACS;FC=FlowCytometry;FPLC=FastProteinLiquidChromatography;GF=GravityFlow;HPLC=HighPerformanceLiquidChromatography;ICC=Immunocytochemistry;IF=Immunofluorescence;IHC=Immunohistochemistry;IP=Immunoprecipitation;NAC=Non-adherentCellAssays;NB=NeutralizationofBioactivity;SE=SandwichELISA;TPE=TargetedProteinExpression;WB=Westernblotting;;AC=AdherentCellAssays;FM=FluorescentMicsroscopy;;;BSC-CM5=BiacoreSensorChipCM5;BSM=BiosactiveSmallMoleculeorPeptide;CDM=CellDifferentiationMedia;;;;;;HealthandFitness;;;DNAExtraction/Purification;;InvivoLikeAssaysSpeciesReactivity:MultipleMammalian
B=Bovine;Ca=Cat;Ch=Chicken;D=Dog;EQ=Equine;GP=GuineaPig;H=Human;M=Mouse;P=Porcine;Pr=Primate;R=Rat;Rb=Rabbit;Y=Yeast;Xe=Xenopus;Ze=Zebrafish;;;;NA-NotApplicable;STP=Step-TactinProteins;AllFormat:AffinityPurified-liquid
mCherryisderivedfromproteinsoriginallyisolatedfromCnidarians(jellyfish,seaanemonesandcorals),andisusedasafluorescenttracerintransfectionandtransgenicexperiments.TheprototypeforthesefluorescentproteinsisGreenFluorescentProtein(GFP),whichisa~27kDaproteinisolatedoriginallyfromthejellyfishAequoriavictoria.ThemCherryproteinisderivedfromDsRed,aredfluorescentproteinrelatedtoGFPisolatedfromso-calleddisccoralsofthegenusDiscosoma.DsRedissimilarinsizeandpropertiestoGFP,but,obviously,producesaredratherthanagreenFluorochrome.
Image:HEK293cellstransfectedinthesamewayandviewedintheconfocalmicroscope.MostHEK293cellsarenottransfectedsoonlythenucleusofthesecellscanbevisualizedwithablueDNAstain.CellswhicharetransfectedwithmCherryarered.StainingwithmCherry(RA22117)isshowninGreen.GreenantibodystainingisonlyseenincellswhichexpressmCherry,asexpected,andthesuperimpositionofthegreenandredsignalsresultsinanorangesignal.Interestingly,strongermCherrystainingisseeninthenucleus,possIBLyduetodegradationofsomemCherrymoleculestoreleasethelowmolecularweightmCherryfluorochrome.ImagecourtesyoftheSemple-RowlandlabattheUniversityofFlorida.
Severalfurthercyclesofmutation,directedmodificationandevolutionaryselectionproducedmCherry,whichhasanexcitationmaximumat587nmandandemissionmaximumat610nm(4).WeexpressedthemCherryproteinsequenceshowninreference4inbacteria,purifiedoutthemCherryandraisedthisrabbitpolyclonalantibody.ThiswasaffinitypurifiedandwasfoundtostainabandoftheexpectedsizeinHEK293cellstransfectedwiththepFin-EF1-mCherryvectordesignedtoexpressmCherry.Asshownbelow,theantibodydoesnotstainanyproteinbandinuntransfectedHEK293cells.
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