Cat#:AGR-043-FLyophilizedPowderLyophilizedPowderThisproductisfreezedried.Allwatermoleculeshavebeenremoved.AntigenIncl.AntigenIncl.ThisantibodyisshippedwithitsantigenFREEofcharge!ShippedatRoomTemp.ShippedatRoomTemp.Thislyophilizedproductisshippedatroomtemperature.Pleaseseeitscertificateofanalysisforfurtherstorageinstructions.Type:PolyclonalSource:RabbitReactivity:h,m,rImmunogenPeptide(C)DYTWNKDNWTFSP,correspondingtoaminoacidresidues72-84ofhumanGPR65(AccessionQ8IYL9).Extracellular,1stloop.Accession(Uniprot)NumberQ8IYL9GeneID8477PeptideconfirmationConfirmedbyaminoacidanalysisandmassspectrometry.HomologyMouse–12/13aminoacidresiduesidentical;rat-11/13aminoacidresiduesidentical.PurityAffinitypurifiedonimmobilizedantigen.FormulationLyophilizedpowder.Reconstitutedantibodycontainsphosphatebufferedsaline(PBS),pH7.4,1%BSA,0.05%NaN3.LabelFluoresceinisothiocyanate(FITC).StoragebeforereconstitutionTheantibodyshipsasalyophilizedpowderatroomtemperature.Uponarrival,itshouldbestoredat-20°C.Reconstitution50µldoubledistilledwater(DDW).Antibodyconcentrationafterreconstitution1mg/ml.StorageafterreconstitutionThereconstitutedsolutioncanbestoredat4°C,protectedfromthelight,forupto1week.Forlongerperiods,smallaliquotsshouldbestoredat-20°C.Avoidmultiplefreezingandthawing.Centrifugeallantibodypreparationsbeforeuse(10000×g5min).ControlantigenstoragebeforereconstitutionLyophilizedpowdercanbestoredintactatroomtemperaturefor2weeks.Forlongerperiods,itshouldbestoredat-20°C.Controlantigenreconstitution100µldoubledistilledwater(DDW).Controlantigenstorageafterreconstitution-20°C.PreadsorptionControl1µgpeptideper1µgantibody.StandardqualitycontrolofeachlotWesternblotanalysis(unlabeledantibody,#AGR-043),anddirectflowcytometry(labeledantibody).Applications:fcMayalsoworkin:ic,lciDirectflowcytometryDirectflowcytometryofliveintacthumanJurkatT-cellleukemiacells:___ Cells.___ Cells+rabbitIgGisotypecontrol-FITC.___ Cells+Anti-GPR65(TDAG8)(extracellular)-FITCAntibody (#AGR-043-F),(5µg).DirectflowcytometryofliveintactmouseM1myeloidleukemiacells:___ Cells.___ Cells+rabbitIgGisotypecontrol-FITC.___ Cells+Anti-GPR65(TDAG8)(extracellular)-FITCAntibody(#AGR-043-F),(5µg).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.Expressionisalsodetected pain relevantlocisuchasthedorsalrootganglianeuronsandparticularlysmalldiameterneuronsresponsIBLefornociception1,2.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-RatLyophilizedPowderControlAntigenIncludedCertificateofAnalysisMSDSLastupdate:20/03/2019Anti-GPR65(TDAG8)(extracellular)Antibody(#AGR-043)isahighlyspecificantibodydirectedagainstanepitopeofthehumanprotein.Theantibodycanbeusedinwesternblot, immunohistochemistry,andlivecellflowcytometry.IthasbeendesignedtorecognizeGPR65fromrat,mouse,andhumansamples.Anti-GPR65(TDAG8)(extracellular)-FITCAntibody(#AGR-043-F)isdirectlyconjugatedtofluoresceinisothiocyanate(FITC).Thislabeledantibodycanbeusedinimmunofluorescentapplicationssuchasdirectlivecellflowcytometry.Forresearchpurposesonly,notforhumanuseDirectflowcytometryDirectflowcytometryofliveintacthumanJurkatT-cellleukemiacells:___ Cells.___ Cells+rabbitIgGisotypecontrol-FITC.___ Cells+Anti-GPR65(TDAG8)(extracellular)-FITCAntibody (#AGR-043-F),(5µg).DirectflowcytometryofliveintactmouseM1myeloidleukemiacells:___ Cells.___ Cells+rabbitIgGisotypecontrol-FITC.___ Cells+Anti-GPR65(TDAG8)(extracellular)-FITCAntibody(#AGR-043-F),(5µg).ReferencesJustus,C.R.etal.(2013)Front.Physiol.4,354.Justus,C.R.etal.(2017)J.Transl.Med.15,204.

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