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 Immobilon -P转印膜   PVDF膜 ·适用于免疫印迹、斑点印迹和其他蛋白质印迹的最佳PVDF膜；·Western Blotting、吸附分析、氨基酸分析、N端蛋白质测序、Dot/Slot blotting、糖蛋白显色和脂多糖分析；·与他相容的染液/染色剂：考马斯亮蓝染色剂、酰胺黑、印度墨水、丽春红、胶体金、CPTS、甲苯胺蓝、透射显影和Sypro Ruby染料. 产品名称Immobilon-P说    明应用于>20KD蛋白质的Western, dot/slot blotting和其他印迹孔    径0.45um尺寸cm×cm26.5×375数    量1卷目录编号IPVH00010 ――――――――――――――――――――――――――――――――――――――― Immobilon - NC膜，混合纤维素酯（Millipore） Immobilon-NC HATF由混合纤维素酯构成，不含细胞生长期间影响细胞完整的表面活性剂。使用这些膜可从事菌落转移实验。Immobilon-NC HAHY由混合纤维素酯构成，但含有转印过程期间能改善可湿性便于操作的表面活性剂。膜的孔径是0.45μm。用于Southern blotting，Northern Blotting，Western blotting，Colony/plaque Lifts，Dot/slot blots，吸附分析，细胞培养.。 产品名称Immobilon - NC说    明应用于Southern杂交， Northern杂交，免疫印迹，结合实验孔    径0.45um尺寸cm×cm33×300数    量1卷目录编号HATF00010