Description
CatalogueNumber	MAB351R
BrandFamily	Chemicon®
TradeName	Chemicon
Description	Anti-GlutamateDecarboxylaseAntibody,65kDaisoform,cloneGAD-6
AlternateNames	GAD65
BackgroundInformation	Gutamicaciddecarboxylase(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.

ProductInformation
Format	Purified
Control	Braintissue
Presentation	Lyophilized.Dissolvecontentsofvialin100µLofsterile,distilledwater.Thisresultsinafinalantibodyconcentrationof1mg/mlin10mMpotassiumphosphate,70mMsodiumchloride,pH7.4containingnopreservatives.

StorageandShippingInformation
StorageConditions	Maintainfor1yearat-20°Cfromdateofshipment.Aliquottoavoidrepeatedfreezingandthawing.Formaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.

Applications
Application	Anti-GlutamateDecarboxylaseAntibody,65kDaisoform,cloneGAD-6isanantibodyagainstGlutamateDecarboxylaseforuseinIH&WB.
KeyApplications	Immunohistochemistry WesternBlotting
ApplicationNotes	Immunohistochemistry:(<=1=""μg/ml)=""optimal=""working=""dilutions=""must=""be=""determined=""by=""end=""user.=""> ImmunohystochemicalStainingProcedures ThefollowingprocedurewasdevelopedtolocalizeGADinratbrainsectionsofcerebellum.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.

BIOLOGicalInformation
Immunogen	PurifiedratbrainGAD.
Clone	GAD-6
Concentration	PleaserefertotheCertificateofAnalysisforthelot-specificconcentration.
Host	Mouse
Specificity	RecognizesthelowermolecularweightisoformofthetwoGADisoformsidentifiedinbrain(Gottlieb,etal.,1986;Chang&Gottlieb,1988).Thismonclonalantibodycanbeusedforimmunohistochemicallocalizationinbrainorpancreas.Anti-GADhasalsobeenusedtolabelpurifiedGADonWesternblots(Chang&Gottlieb,1988).
Isotype	IgG2a
SpeciesReactivity	Human Rat
AntibodyType	MonoclonalAntibody
EntrezGeneNumber	NM_000818.1
EntrezGeneSummary	Thisgeneencodesoneofseveralformsofglutamicaciddecarboxylase,identifiedasamajorautoantigenininsulin-dependentdiabetes.Theenzymeencodedisresponsibleforcatalyzingtheproductionofgamma-aminobutyricacidfromL-glutamicacid.ApathogenicroleforthisenzymehasbeenidentifiedinthehumanpancreassinceithasbeenidentifiedasanautoantibodyandanautoreactiveTcelltargetininsulin-dependentdiabetes.Thisgenemayalsoplayaroleinthestiffmansyndrome.
GeneSymbol	GAD2 MGC161607 GAD65 MGC161605 GAD-65 EC4.1.1.15
PurificationMethod	AmmoniumsulfateprecipitationandDEAE-cellulosechromatography
UniProtNumber	Q05329
UniProtSummary	FUNCTION:SwissProt:Q05329#CatalyzestheproductionofGABA. COFACTOR:Pyridoxalphosphate. SIZE:585aminoacids;65411Da SUBUNIT: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.
MolecularWeight	65kDa

PhysicochemicalInformation

Dimensions

MaterialsInformation

MaterialsInformation

Millipore 提供三种不同的 Immobilon PVDF 膜，是针对不同的蛋白质印迹应用的理想选择。 现在我们还 提供采用按规格裁切的印迹方片膜和两层过滤纸组成的印迹三明治。   与硝酸纤维素膜相比，Immobilon 膜具有多种优点。 它们不易破裂或卷 曲，便于裁剪且在切割过程中不会碎裂。 它们还有低背景、广泛溶剂相容性以及优异的着色能力。 此外，它们可以重复检测。 Immobilon-P 膜（沃比森-供应） ·         0.45μm 孔径 ·         建议用于大多数免疫印迹，特别在蛋 白质大于 20kDa 时 ·         Millipore 的快速免疫检测实验方法不需要膜封闭并缩短洗涤时间，将检测时间至少减少了两个小时，而不致降低灵敏 度或特异性 用于高通量实验室的印迹三明治 ·         当您需要处理多个印迹时，印迹三明治是一种便利省时的选择。 我们的印迹三明 治包含多片 Immobilon-P 膜，其中插入了按规格裁切的多张层析级印迹纸。 转印滤纸 ·         按照大多数免疫印迹尺寸进行裁切的层析级转印滤纸。 Immobilon-PSQ 膜（沃比森-供应） ·         0.2μm 孔径和大的内表面面积 ·         较其它膜具有更高的 蛋白质吸附率和测序得率 ·         建议用于小于 20kDa 的印迹蛋白质 ·         防止小分子量的蛋白质的渗漏 Immobilon-FL 膜（沃比森-供应） ·        针对荧光应用进行优化的转印膜 ·         极低的背景提高了所 有荧光检测实验的灵敏度 ·         与所有常用的荧光探针（所有激发和发射波长）兼容 ·         是多态性检测和化学荧光应用的理想选择