DescriptionCatalogueNumberMAB351RBrandFamilyChemicon®TradeNameChemiconDescriptionAnti-GlutamateDecarboxylaseAntibody,65kDaisoform,cloneGAD-6AlternateNamesGAD65BackgroundInformationGutamicaciddecarboxylase(GAD;E.C.4.1.1.15)istheenzymeresponsIBLefortheconversionofglutamicacidtogamma-aminobutyricacid(GABA),themajorinhibitorytransmitterinhigherbrainregions,andputativeparacrinehormoneinpancreaticislets.TwomolecularformsofGAD(65kDaand67kDa,64%aaidentitybetweenforms)arehighlyconservedandbothformsareexpressedintheCNS,pancreaticisletcells,testis,oviductandovary.Theisoformsareregionallydistributedcytoplasmicallyinthebrainsofratsandmice(Sheikh,S.etal.1999).GAD65isanampiphilic,membrane-anchoredprotein(585aa),encodedonhumanchromosome10,andisresponsibleforvesicularGABAproduction.GAD67iscytoplasmic(594aa.),encodedonchromosome2,andseemstoberesponsibleforsignificantcytoplasmicGABAproduction.GADexpressionchangesduringneuraldevelopmentinratspinalcord.GAD65isexpressedtransientlyincommissuralaxonsaroundE13butisdownregulatedthenextdaywhileGAD67expressionincreasesmostlyinthesomataofthoseneurons(Phelps,P.etal.1999).Inmatureratpancreas,GAD65andGAD67appeartobedifferentiallylocalized,GAD65primarilyininsulin-containingbetacellsandGAD67inglucagon-containing(A)cells(Li,L.etal.1995).GAD67expressionseemstobeparticularlyplasticandcanchangeinresponsetoexperimentalmanipulation(forexampleneuronalstimulationortransection)ordiseaseprogressionandemergentdisorderslikeschizophrenia(Volketal.,2000).ColocalizationofthetwoGADisoformsalsoshowschangesinGAD65/GAD67distributionscorrelatedwithcertaindiseasestatessuchasIDDMandSMS.ProductInformationFormatPurifiedControlBraintissuePresentationLyophilized.Dissolvecontentsofvialin100µLofsterile,distilledwater.Thisresultsinafinalantibodyconcentrationof1mg/mlin10mMpotassiumphosphate,70mMsodiumchloride,pH7.4containingnopreservatives.StorageandShippingInformationStorageConditionsMaintainfor1yearat-20°Cfromdateofshipment.Aliquottoavoidrepeatedfreezingandthawing.Formaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.ApplicationsApplicationAnti-GlutamateDecarboxylaseAntibody,65kDaisoform,cloneGAD-6isanantibodyagainstGlutamateDecarboxylaseforuseinIH&WB.KeyApplicationsImmunohistochemistryWesternBlottingApplicationNotesImmunohistochemistry:(ImmunohystochemicalStainingProceduresThefollowingprocedurewasdevelopedtolocalizeGADinratbrainsectionsofcerebellum.Performallstepsatroomtemperatureunlessotherwiseindicated.Wherenormalserumisindicated,usenormalserumfromthesamespeciesasthesourceofthesecondaryantibody.Thisprocedurerepresentssuggestedguidelinesfortheuseofanti-GAD.Fixationregimen,antibodyconcentrations,andincubationconditionsforagivenexperimentalsystemshouldbedeterminedempirically.1.Perfuseratswith100mMphosphatebuffer,pH7.4,containing1%paraformaldehyde,0.34%L-lysine,and0.05%sodiumm-periodate(1%PLP).2.Postfixbrainsin1%PLPfor1-2hours.Longerfixationtimesmayreducelabelingintensity.3.Transferbrainsto100mMphosphatebuffercontaining30%sucrose,andgentlyagitateonashakerplatformat+4°Cfor48-60hours.4.Usingaslidingmicrotome,cut30mmsectionsoffrozencerebellum.Asthesectionsarecut,collecttheminavialofcold100mMphosphatebuffer.5.Incubatesectionsinphosphate-bufferedsaline(PBS)containing1.5%normalserumand0.2%TritonX-100for30minutes.6.Onashakerplatform,incubatesectionswithanti-GAD(dilutedinPBScontaining1.5%normalserumand0.2%TritonX-100toafinalantibodyconcentrationof1mg/ml)for12-36hoursat+4°C.7.Onashakerplatform,rinsesectionseighttimes,10-15minutesperrinse,inPBS.8.Detectwithastandardsecondaryantibodydetectionsystem(Hsuetal.,1981;Falini&Taylor,1983;Harlow&Lane,1988;Taylor,1978).9.Mountsections,dehydrate,andapplycoverslips.BIOLOGicalInformationImmunogenPurifiedratbrainGAD.CloneGAD-6ConcentrationPleaserefertotheCertificateofAnalysisforthelot-specificconcentration.HostMouseSpecificityRecognizesthelowermolecularweightisoformofthetwoGADisoformsidentifiedinbrain(Gottlieb,etal.,1986;Chang&Gottlieb,1988).Thismonclonalantibodycanbeusedforimmunohistochemicallocalizationinbrainorpancreas.Anti-GADhasalsobeenusedtolabelpurifiedGADonWesternblots(Chang&Gottlieb,1988).IsotypeIgG2aSpeciesReactivityHumanRatAntibodyTypeMonoclonalAntibodyEntrezGeneNumberNM_000818.1EntrezGeneSummaryThisgeneencodesoneofseveralformsofglutamicaciddecarboxylase,identifiedasamajorautoantigenininsulin-dependentdiabetes.Theenzymeencodedisresponsibleforcatalyzingtheproductionofgamma-aminobutyricacidfromL-glutamicacid.ApathogenicroleforthisenzymehasbeenidentifiedinthehumanpancreassinceithasbeenidentifiedasanautoantibodyandanautoreactiveTcelltargetininsulin-dependentdiabetes.Thisgenemayalsoplayaroleinthestiffmansyndrome.GeneSymbolGAD2MGC161607GAD65MGC161605GAD-65EC4.1.1.15PurificationMethodAmmoniumsulfateprecipitationandDEAE-cellulosechromatographyUniProtNumberQ05329UniProtSummaryFUNCTION:SwissProt:Q05329#CatalyzestheproductionofGABA.COFACTOR:Pyridoxalphosphate.SIZE:585aminoacids;65411DaSUBUNIT:Homodimer(Bysimilarity).SUBCELLULARLOCATION:Cytoplasm,cytosol.Cytoplasmicvesicle.Celljunction,synapse,presynapticcellmembrane;Lipid-anchor.Golgiapparatusmembrane;Peripheralmembraneprotein;Cytoplasmicside.Note=Associatedtocytoplasmicvesicles.Inneurons,cytosolicleafletofGolgimembranesandpresynapticclusters.PTM:Phosphorylated;whichdoesnotaffectkineticparametersorsubcellularlocation.&Palmitoylated;whichisrequiredforpresynapticclustering.SIMILARITY:SwissProt:Q05329##BelongstothegroupIIdecarboxylasefamily.MolecularWeight65kDaPhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation