Detectsabandofthepropermolecularweight,30kD,inlysatesfromE.coliexpressingGFP,butnotinE.colithatdonotexpressorinlysatespreparedfromE.colicontainingtheGFP-expressingplasmidbutnotinduced,detectGFPinlysatesfromCOSorprimaryneuronsorfibroblastcellstransfectedwithplasmidsdirectingexpressionGFPoratau-GFPfusionprotein.Didnotdetectanysignalwhen50ugofproteinfromaratbrainlysate(negativecontrol)wasfractionatedontheSDS-PAGE.
Applications:|SuitableforuseinWesternBlot,ImmunocytochemistryandImmunoprecipitation.Otherapplicationshavenotbeentested.
RecommendedDilutions
Immunocytochemistry:Cellswerefixedusing4%formaldehydeinPBS,pH7.4for20minutesatRT.Thecellsareperme
ABIlizedwith0.2%TritonX-100and2%serum(correspondingtotheanimalinwhichthesecondaryantibodywasraised)inPBSfor20minutesatRT.CellculturesarethenincubatedwithprimaryantiserainPBScontaining2%serum(asabove),at4°C,overnight.Hasalsobeenusedsuccessfullywiththeavidin/biotin/peroxidasedetectionsystem.Cultureswerewashedfourtimes(10minuteseach)betweensteps,andreactionproductisdevelopedwitha0.05%diaminobenzidine/0.02%hydrogenperoxidesolutiononicefor8-10minutes.Optimaldilutionstobedeterminedbytheresearcher.
StorageandStability
Storeproductat4°Ciftobeusedimmediatelywithintwoweeks.Forlong-termstorage,aliquottoavoidrepeatedfreezingandthawingandstoreat-20°C.Aliquotsarestableat-20°Cfor12monthsafterreceipt.Diluterequiredamountonlypriortoimmediateuse.Furtherdilutionscanbemadeinassaybuffer.Caution:FITCconjugatesaresensitivetolight.Formaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.
Note:Applicationsarebasedonunconjugatedantibody.
References
1.Hallam,S.J.,etal.,NatureNeuroscience5:1137-1146(2002).2.Sippy,T.,etal.,NatureNeuroscience10:1031-1038(2003).3.Jakobsson,J.,etal.,EuropeanJournalofNeuroscience19:761-765(2004).4.Zhao,S.,etal.,EuropeanJournalofNeuroscience19:1133-1140(2004).5.Yawata,S.,etal.,JournalofNeuroscience26:3626-3633(2006).