Description | |
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CatalogueNumber | 17-472 |
BrandFamily | Upstate |
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Description | Phospho-GSK-3β(Ser9)STARELISAKit |
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BackgroundInformation | I.TESTPRINCIPLETheUPSTATE®colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheGSK-3βplateiscoatedwithaspecificmousemonoclonalGSK-3&betacaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingGSK-3βantigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-GSK-3βantibodytodetectthecapturedphosphor-GSK-3β(Ser9)ontheplatewell.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardlineofdetection. II.GSK-3βBACKGROUNDGlycogenSynthaseKinase-3differsfrommostserine/threoninekinasesinthatitisactiveintheabsenceoftheactionofsignalingpathways.Whenitbecomesphosphorylated,theproteinbecomesinactive.Therearetwoisoformsthatexist,GSK-3αandGSK-3β.ThefunctionofGSK-3istophosphorylateGlycogenSynthaseandtherebyinactivateit.InsulinactionstimulatesthePI3KinasepathwaythatresultsintheactivationofAkt.AktphosphorylatesGSK-3,therebyinactivatingit.GlycogenSynthase,GSK-3"sdownstreamsubstrate,isthenrapidlydephosphorylatedandactivated.OtherGSK-3substratesincludeJun(oninhibitorysites),andeIF2B.DetectionofGSK-3"sphosphorylationstatusoftheAktsite(Ser21)onGSK-3issuitableforsurrogateassaysoftheactivationstateofthepathway. |
MaterialsRequiredbutNotDelivered | 1.Multi-channelorrepeatingPipettes 2.Plateshaker(optional) 3.Pipettorsandtipscapableofaccuratelymeasuring1-1000µL 4.GraduatedSEROlogicalpipettes 5.96-wellmicrotiterPlateReaderwith450nmfilter 6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata 7.Microfugetubesforstandardandsampledilutions 8.Mechanicalvortex 9.1litercontainer 10.Distilledordeionizedwater |
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Detectionmethod | Colorimetric |
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StorageConditions | Maintaintheunopenedkitat2-8°Cuntilexpirationdate. Precautions •Theinstructionsprovidedhavebeendesignedtooptimizethekit"sperformance.Deviationfromtheinstructionsmayresultinsuboptimalperformanceofthekitandthefailuretoproduceaccuratedata. •CausticMaterial:StopSolution.Caution:Eye,hand,face,andclothingprotectionshouldbewornwhenhandlingthismaterial. •SafetyWarningsandPrecautions:Thiskitisdesignedforresearchuseonlyandnotrecommendedforinternaluseinhumansoranimals.Allchemicalsshouldbeconsideredpotentiallyhazardousandprinciplesofgoodlaboratorypracticeshouldbefollowed. •TheDetectionAntibodyandELISADiluentcontainsodiumazide.Sodiumazidemayreactwithcopperandleadplumbingtoformhighlyexplosivemetalazides.Upondisposal,flushwithlargeamountsofwatertopreventazidebuild-up.Avoidcontactwithskin. •TheAnti-RabbitIgGHRPConjugateandHRPDiluentcontainthimerosal.Thimerosalishighlytoxicbyinhalation,contactwithskinorifswallowed.ThimerosalisapossIBLemutagenandshouldbehandledaccordingly. |
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Application | ThisPhospho-GSK-3β(Ser9)STARELISAKitisusedtomeasure&quantifyGSK-3βlevels. |
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EntrezGeneSummary | Glycogensynthasekinase-3(GSK3)isaproline-directedserine-threoninekinasethatwasinitiallyidentifiedasaphosphorylatingandinactivatingglycogensynthase.Twoisoforms,alpha(GSK3A;MIM606784)andbeta,showahighdegreeofaminoacidhomology(StambolicandWoodgett,1994[PubMed7980435]).GSK3Bisinvolvedinenergymetabolism,neuronalcelldevelopment,andbodypatternformation(Plyteetal.,1992[PubMed1333807]).[suppliedbyOMIM] |
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UniProtSummary | FUNCTION:SwissProt:P49841#ParticipatesintheWntsignalingpathway.Implicatedinthehormonalcontrolofseveralregulatoryproteinsincludingglycogensynthase,MYBandthetranscriptionfactorJUN.PhosphorylatesJUNatsitesproximaltoitsDNA-bindingdomain,therebyreducingitsaffinityforDNA. SIZE:420aminoacids;46744Da SUBUNIT:Monomer(Bysimilarity).InteractswithNINandCABYR. TISSUESPECIFICITY:Expressedintestis,thymus,prostateandovaryandweaklyexpressedinlung,brainandkidney. PTM:PhosphorylatedbyAKT1andILK1. SIMILARITY:SwissProt:P49841##Belongstotheproteinkinasesuperfamily.CMGCSer/Thrproteinkinasefamily.GSK-3subfamily.&Contains1proteinkinasedomain. |
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