Cat#:AGR-043LyophilizedPowderLyophilizedPowderThisproductisfreezedried.Allwatermoleculeshavebeenremoved.AntigenIncl.AntigenIncl.ThisantibodyisshippedwithitsantigenFREEofcharge!ShippedatRoomTemp.ShippedatRoomTemp.Thislyophilizedproductisshippedatroomtemperature.Pleaseseeitscertificateofanalysisforfurtherstorageinstructions.Type:PolyclonalSource:RabbitReactivity:h,m,rImmunogenPeptide(C)DYTWNKDNWTFSP,correspondingtoaminoacidresidues72-84ofhumanGPR65(AccessionQ8IYL9).1stextracellularloop.Accession(Uniprot)NumberQ8IYL9GeneID8477PeptideconfirmationConfirmedbyaminoacidanalysisandmassspectrometry.HomologyMouse–12/13aminoacidresiduesidentical;rat-11/13aminoacidresiduesidentical.PurityAffinitypurifiedonimmobilizedantigen.FormulationLyophilizedpowder.Reconstitutedantibodycontainsphosphatebufferedsaline(PBS),pH7.4,1%BSA,0.05%NaN3.StoragebeforereconstitutionTheantibodyshipsasalyophilizedpowderatroomtemperature.Uponarrival,itshouldbestoredat-20°C.Reconstitution25µl,50µlor0.2mldoubledistilledwater(DDW),dependingonthesamplesize.Antibodyconcentrationafterreconstitution0.8mg/ml.StorageafterreconstitutionThereconstitutedsolutioncanbestoredat4°Cforupto1week.Forlongerperiods,smallaliquotsshouldbestoredat-20°C.Avoidmultiplefreezingandthawing.Centrifugeallantibodypreparationsbeforeuse(10000xg5min).ControlantigenstoragebeforereconstitutionLyophilizedpowdercanbestoredintactatroomtemperaturefor2weeks.Forlongerperiods,itshouldbestoredat-20°C.Controlantigenreconstitution100µldoubledistilledwater(DDW).Controlantigenstorageafterreconstitution-20°C.PreadsorptionControl1µgpeptideper1µgantibody.StandardqualitycontrolofeachlotWesternblotanalysis.Applications:ifc,ih,wbMayalsoworkin:ic,ip,lciWesternblotWesternblotanalysisofhumanJurkatT-cellleukemia(lanes1and4),humanTHP-1monocyticleukemia(lanes2and5)andhumanHL-60promyelocyticleukemia(lanes3and6)celllinelysates:1-3. Anti-GPR65(TDAG8)(extracellular) Antibody(#AGR-043),(1:200).4-6.Anti-GPR65(TDAG8)(extracellular)Antibody,preincubatedwiththecontrolpeptideantigen.Westernblotanalysisofrat(lanes1and3)andmouse(lanes2and4) brainmembranes:1,2. Anti-GPR65(TDAG8)(extracellular) Antibody(#AGR-043),(1:200).3,4.Anti-GPR65(TDAG8)(extracellular)Antibody,preincubatedwiththecontrolpeptideantigen.ImmunohistochemistryExpressionofGPR65inratfornixImmunohistochemicalstainingofimmersion-fixed,freefloatingratfrozenbrainsectionsusing Anti-GPR65(TDAG8)(extracellular) Antibody(#AGR-043),(1:1200),followedbygoat-anti-rabbit-AlexaFluor-488.GPR65staining(green)appearsinglialprocesses(verticalarrows)andinthesideoffornix(FX)facingtheventricle(V,horizontalarrow).CellnucleiarestainedwithDAPI(blue).IndirectflowcytometryIndirectflowcytometryofliveintacthumanJurkatT-cellleukemiacells:___ Cells.___ Cells+goat-anti-rabbit-FITC.___ Cells+ Anti-GPR65(TDAG8)(extracellular) Antibody(#AGR-043),5µg+goat-anti-rabbit-FITC.ReferencesJustus,C.R.etal.(2013)Front.Physiol.4,354.Justus,C.R.etal.(2017)J.Transl.Med.15,204.ScientificbackgroundGPR65,alsocalledTDAG8(T-celldeathassociatedgene8protein),isapH-sensingG-proteincoupledreceptor(GPCR)activatedbyacidicextracellularpHthroughtheprotonationofseveralhistidineresiduesinthereceptor’ssequence.GPR65isinvolvedincancercellmetastasisandproliferation,immunecellfunction,inflammation,andbloodvesselformation1.HumanGPR65genehasbeenmappedtoalocationthatisassociatedwithTcelllymphomaandleukemiaabnormalities.GPR65isprimarilyexpressedinimmunecellsandleukocyte-richtissuessuchascirculatingperipheralleukocytes,spleen,thymus,andtonsils.ExpressionisalsodetectedpainrelevantlocisuchasthedorsalrootganglianeuronsandparticularlysmalldiameterneuronsresponsIBLefornociception1,2.GPR65hastheABIlitytoaffecttumordevelopmentandgrowth.OverexpressionofGPR65inLewislungcarcinomacellsincreasestumorgrowthinmiceandmayfacilitateresistancetoacidosis-mediatedcelldeath invitro throughproteinkinaseA(PKA)andERKrelatedpathways1.KnockdownofGPR65inNCI-H460humannon-smallcelllungcancercellsdecreasescellsurvivalinacidiccondition.AnotherstudyshowsthatactivationofGPR65byacidosiscanpromoteevasionofcellapoptosisunderglutaminestarvationanditsoverexpressionhasbeenreportedtotransformimmortalizedmammaryepithelialcells1.Applicationkey:CBE-Cell-basedELISA,FC-Flowcytometry,ICC-Immunocytochemistry,IE-IndirectELISA,IFC-Indirectflowcytometry,IHC-Immunohistochemistry,IP-Immunoprecipitation,LCI-Livecellimaging,N-Neutralization,WB-WesternblotSpeciesreactivitykey:H-Human,M-Mouse,R-RatLyophilizedPowderControlAntigenIncludedClickheretoreceivea25µlfreetrialsample!CertificateofAnalysisMSDSLastupdate:11/12/2018Anti-GPR65(TDAG8)(extracellular)Antibody(#AGR-043)isahighlyspecificantibodydirectedagainstanepitopeofthehumanprotein.Theantibodycanbeusedinwesternblot,immunohistochemistry,andlivecellflowcytometry.IthasbeendesignedtorecognizeGPR65fromrat,mouse,andhumansamples.Forresearchpurposesonly,notforhumanuseWesternblotWesternblotanalysisofhumanJurkatT-cellleukemia(lanes1and4),humanTHP-1monocyticleukemia(lanes2and5)andhumanHL-60promyelocyticleukemia(lanes3and6)celllinelysates:1-3. Anti-GPR65(TDAG8)(extracellular) Antibody(#AGR-043),(1:200).4-6.Anti-GPR65(TDAG8)(extracellular)Antibody,preincubatedwiththecontrolpeptideantigen.Westernblotanalysisofrat(lanes1and3)andmouse(lanes2and4) brainmembranes:1,2. Anti-GPR65(TDAG8)(extracellular) Antibody(#AGR-043),(1:200).3,4.Anti-GPR65(TDAG8)(extracellular)Antibody,preincubatedwiththecontrolpeptideantigen.

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