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Millipore/17-477 | PRAS40 STAR ELISA Kit/17-477/96 assays
  • Millipore/17-477 | PRAS40 STAR ELISA Kit/17-477/96 assays

Millipore/17-477 | PRAS40 STAR ELISA Kit/17-477/96 assays

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货号: 17-477
品牌: Millipore
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    • Description
      CatalogueNumber17-477
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      DescriptionPRAS40STARELISAKit
      AlternateNames
      • SignalTranscductionAssayReactionPRAS40ELISA
      BackgroundInformationI.TESTPRINCIPLE
      TheUPSTATE®colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.ThePRAS40plateiscoatedwithaspecificmousemonoclonalPRAS40captureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingPRAS40antigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanyunboundnon-specificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-phospho-PRAS40(Thr246)antibodytodetectthecapturedPRAS40ontheplatewellthatisphosphorylatedonThr246.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Thisallowsforasensitiveenzymaticdetectionofthesample.
      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.ThekitalsoincludesastandardthatisrunasbothapositivecontrolandtogenerateastandardcurveforphosphorylatedPRAS40(Thr246)measurement.

      II.PRAS40BACKGROUND
      PRAS40(ProlineRichAktSubstrate,40kDa),alsoknownasAKT1S1,isaubiquitouslyexpressproteinthatisphosphorylatedonresidueThr246viathePI3K/Aktpathway.ItwasoriginallydiscoveredasanAktsubstrateasithastheputativeAktphosphorylationmotifofRxRxxpS/pT2.Thisphosphorylationallowsittobind14-3-3andRaptorofthemTORcomplexmTORC1.PRAS40isknowntobindtoandregulatemTORC1(mTOR,Raptor,mLST8)kinasesactivitythatisactivatedbyinsulindownstreamofPI3KandAktandsubsequentlyphosphorylatesp70S6Kand4EBP1.PRAS40isknowntohaveaputativeTOSmotif(FVMDE)thatallowsitbindtoRaptorofmTORC1intheabsenceofinsulin.ItisthoughtthatthroughitbindingtotheRaptor,PRAS40inhibitsthekinaseactivityofmTORC1bypreventingtobindtoitssubstratessuchasp70S6Kand4EBP1.
      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
      • 1.CapturePlatepre-coatedwithanti-PRAS40antibody:(PartNo.17-477A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • 2.Anti-PRAS40detectionantibody:(PartNo.17-477B)Onebottle(11mL)ofantiPRAS40detectionantibodycontainingsodiumazide,readytouse.
      • 3.ELISADiluent:(PartNo.17-477C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse
      • 4.25XELISAWashBuffer:(PartNo.17-477D)Onebottle(50mL)of25XELISAWashBuffer.
      • 5.Anti-RabbitIgGHRPconjugate:(PartNo.17-477E)Onevial(125μL)of100Xanti-rabbitHRPconjugate.
      • 6.HRPDiluent:(PartNo.17-477F)Onebottle(25mL)ofHRPDiluent
      • 7.TMBSolution:(PartNo.17-477G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • 8.StopSolution:(PartNo.17-477H)Onebottle(25mL)ofstopsolution,readytouse.
      • 9.PRAS40Standard:(PartNo.17-477I)TwovialsofPRAS40standard,lyophilized.
      • 10.PlateCovers:Twoplatecovers.
      DetectionmethodColorimetric
      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
      ApplicationThePRAS40colorimetricSTARELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtoquantifyspecificlevelsofsignalingtargetsindenaturedcellextracts.
      KeyApplications
      • ELISA
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      AnalytesAvailable
      • PRAS40
      EntrezGeneNumber
      EntrezGeneSummaryAKT1S1isaproline-richsubstrateofAKT(MIM164730)thatbinds14-3-3protein(seeYWHAH,MIM113508)whenphosphorylated(Kovacinaetal.,2003[PubMed12524439]).[suppliedbyOMIM]
      GeneSymbol
      • AKT1S1
      • Lobe
      • MGC2865
      • PRAS40
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:Q96B36#Mayplayanimportantroleinphosphatidylinositol3-kinase(PI3K)-AKT1survivalsignaling.SubstrateforAKT1phosphorylation,butcanalsobeactivatedbyAKT1-independentmechanisms.Itsroleinsurvivalsignalingpathwaysmaybemodulatedbyoxidativestress.Mayalsoplayaroleinnervegrowthfactor-mediatedneuroprotection.
      SIZE:256aminoacids;27383Da
      SUBUNIT:Thephosphorylatedforminteractswith14-3-3.
      SUBCELLULARLOCATION:Cytoplasm,cytosol(Bysimilarity).Note=Foundinthecytosolicfractionofthebrain(Bysimilarity).
      TISSUESPECIFICITY:Widelyexpressedwithhighestlevelsofexpressioninliverandheart.Expressedathigherlevelsincancercelllines(e.g.A549andHeLa)thaninnormalcelllines(e.g.HEK293).
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:15.6pg/mL
        RangeofDetection:31.2to2000pg/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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