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Millipore/17-459 | phospho-IRS1 (Ser307 mouse/ Ser312 Human) STAR ELISA Kit/17-459/96 assays
  • Millipore/17-459 | phospho-IRS1 (Ser307 mouse/ Ser312 Human) STAR ELISA Kit/17-459/96 assays

Millipore/17-459 | phospho-IRS1 (Ser307 mouse/ Ser312 Human) STAR ELISA Kit/17-459/96 assays

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貨號: 17-459
品牌: Millipore
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    • Description
      CatalogueNumber17-459
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      Descriptionphospho-IRS1(Ser307mouse/Ser312Human)STARELISAKit
      AlternateNames
      • InsulinReceptorSubstrate
      BackgroundInformationI.TESTPRINCIPLE
      TheUPSTATE?colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheIRS1plateiscoatedwithaspecificmousemonoclonalcaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingIRS1antigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanyunboundnon-specificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-phospho-IRS1(Ser312)antibodytodetectthecapturedIRS1ontheplatewellthatisphosphorylatedonSer312.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.
      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardcurve.

      II.IRS1BACKGROUND
      IRS(InsulinReceptorSubstrate)proteinsaretheeffectorsofbothInsulinandIGF-initiatedsignaling.Insulin,inadditiontoworkingverysimilarlytoagrowthfactorinstimulatingreceptortyrosinekinases,isessentialformaintainingglucosehomeostasisandregulatingcarbohydrate,lipid,andproteinmetabolism.Thesignalingeventsofinsulinaremediatedviaavastandhighlyintegratednetworkofmoleculesthatcontrolsseveralprocesses.Thereare4IRSproteins,1-4.Itisbelievedthattheseproteinsservecomplementaryfunctionsindifferenttissues.ThetwoprominentplayersareIRS1andIRS2withIRS1playingimportantrolesinskeletalmuscleandIRS2intheliver.TheIRSproteinslinktheinsulinreceptorwithmanysignalingpathwaysincludingthePI3Kinase/Akt/mTORandtheMAPKinasepathways.ThePI3K/Akt/mTORpathwayisinvolvedwithmanymetabolicactionsofinsulinsignaling.MAPKisinvolvedintheregulationofsomegenesandworkswithPI3Kinasetocontrolcellgrowthanddifferentiation.InadditiontotheinsulinreceptortyrosinekinaseactivityintypeIIdiabetespatientsandmice,studieshaveshownagreaterdefectincomponentsoftheinsulinsignalingpathwayincludingInsulinReceptorSubstrate(IRS)phosphorylationortheactivationofPhosphoinositide3-Kinase(PI3Kinase).Insulinstimulatesthesignalingcascadebybindingtotheinsulinreceptorthroughitsextracellularαsubunits.Thisstimulatesthetyrosinekinaseactivityoftheβsubunitsofthereceptor.ThereceptorthenautophosphorylatesitselfandphosphorylatestheIRS(InsulinReceptorSubstrate)proteinsthatareveryimportantmodulatorsofinsulinsignaling.IRSbindstothephosphorylatedtyrosinesoftheβsubunitoftheinsulinreceptor.TheysharePHandPTBdomainsneartheirN-termini,andmultipleTyrphosphorylationmotifsintheirC-terminalregions.Proteinswhichbindtotyrosine-phosphorylatedIRS-proteinsincludePI3Kinasep85,GRB2,SHP2,Nck,Crk,andFyn.IRS1appearstobeprincipallyinvolvedinIGF-signalingandcytoskeletalgrowth.IRS2appearstobethecriticalmediatorofInsulinsignaling,asgeneticablationresultsintypeIIdiabetes.IRS3isexpressedprimarilyinADIpocytesandisanunusuallypotentactivatorofPI3Kinase.IRS4lacksthetyrosineresidueswhichintheotherIRS-proteinsbindstoSHP2.
      MaterialsRequiredbutNotDelivered1.Multi-channelorrepeatingPipettes
      2.Plateshaker(optional)
      3.Pipettorsandtipscapableofaccuratelymeasuring1-1000μL
      4.GraduatedSEROlogicalpipettes
      5.96-wellmicrotiterPlateReaderwith450nmfilter
      6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata
      7.Microfugetubesforstandardandsampledilutions
      8.Mechanicalvortex
      9.1litercontainer
      10.Distilledordeionizedwater
      ProductInformation
      Components
      • CapturePlatepre-coatedwithanti-IRS1antibody:(PartNo.17-459A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • Anti-phospho-IRS1(Ser312)detectionantibody:(PartNo.17-459B)Onebottle(11mL)ofanti-phospho-IRS1(Ser312)detectionantibodycontainingsodiumazide,readytouse.
      • ELISADiluent:(PartNo.17-459C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 25XELISAWashBuffer:(PartNo.17-459D)Onebottle(50mL)of25XELISAWashBuffer.
      • Anti-RabbitIgGHRPconjugate:(PartNo.17-459E)Onevial(125μL)of100Xanti-rabbitHRPconjugatecontainingthimerosal.
      • HRPDiluent:(PartNo.17-459F)Onebottle(25mL)ofHRPDiluentcontainingthimerosal.
      • TMBSolution:(PartNo.17-459G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • StopSolution:(PartNo.17-459H)Onebottle(25mL)ofstopsolution,readytouse.
      • Phospho-IRS1(Ser312)Standard:(PartNo.17-459I)TwovialsofIRS1standard,lyophilized.
      • PlateCovers:Twoplatecovers.
      DetectionmethodChromogenic
      StorageandShippingInformation
      StorageConditionsMaintaintheunopenedkitat2-8°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.
      Applications
      ApplicationThisphospho-IRS1(Ser307mouse/Ser312Human)STARELISAKitisusedtomeasure&quantifyIRS1levels.
      KeyApplications
      • ELISA
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      • Mouse
      • Rat
      AnalytesAvailable
      • IRS1
      EntrezGeneNumber
      GeneSymbol
      • IRS1
      • IRS-1
      • HIRS-1
      Modifications
      • Phosphorylation
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:P35568#Maymediatethecontrolofvariouscellularprocessesbyinsulin.WhenphosphorylatedbytheinsulinreceptorbindsspecificallytovariouscellularproteinscontainingSH2domainssuchasphosphatidylinositol3-kinasep85subunitorGRB2.Activatesphosphatidylinositol3-kinasewhenboundtotheregulatoryp85subunit(Bysimilarity).
      SIZE:1242aminoacids;131591Da
      SUBUNIT:InteractswiththeNPXYmotifoftyrosine-phosphorylatedIGF1RandINSRviathePTBdomain.Bindstophosphatidylinositol3-kinasep85subunitviathephosphorylatedYXXMmotifs.BindsROCK1.BindstoUBTFandPIK3CAinnuclearextracts(Bysimilarity).InteractswithSOCS7.
      PTM:SerinephosphorylationofIRS1isamechanismforinsulinresistance.Ser-312phosphorylationinhibitsinsulinactionthroughdisruptionofIRS1interactionwiththeinsulinreceptor(Bysimilarity).&PhosphorylationofTyr-896isrequiredforGRB2-binding(Bysimilarity).DISEASE:SwissProt:P35568#PolymorphismsinIRS1maybeinvolvedintheetiologyofnon-insulin-dependentdiabetesmellitus(NIDDM)[MIM:125853].
      SIMILARITY:SwissProt:P35568##Contains1IRS-typePTBdomain.&Contains1PHdomain.
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:1.5Units/mL
        RangeofDetection:1.6to100Units/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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