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Millipore/17-478 | phospho-PRAS40 (Thr246) STAR ELISA Kit/17-478/96 assays
  • Millipore/17-478 | phospho-PRAS40 (Thr246) STAR ELISA Kit/17-478/96 assays

Millipore/17-478 | phospho-PRAS40 (Thr246) STAR ELISA Kit/17-478/96 assays

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貨號(hào): 17-478
品牌: Millipore
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    • Description
      CatalogueNumber17-478
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      Descriptionphospho-PRAS40(Thr246)STARELISAKit
      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)thatallowsitbindtoRaptorofmTORC1intheabsenceofinsulin1.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.17-478A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • 2.Anti-phospho-PRAS40(Thr246)detectionantibody:(PartNo.17-478B)Onebottle(11mL)ofanti-phospho-PRAS40(Thr246)detectionantibodycontainingsodiumazide,readytouse.
      • 3.ELISADiluent:(PartNo.17-478C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 5.Anti-RabbitIgGHRPconjugate:(PartNo.17-478E)Onevial(125μL)of100Xanti-rabbitHRPconjugate.
      • 6.HRPDiluent:(PartNo.17-478F)Onebottle(25mL)ofHRPDiluent.
      • 7.TMBSolution:(PartNo.17-478G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse
      • 8.StopSolution:(PartNo.17-478H)Onebottle(25mL)ofstopsolution,readytouse.
      • 9.Phospho-PRAS40(Thr246)Standard:(PartNo.17-478I)TwovialsofphosphorylatedPRAS40(Thr246)standard,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
      ApplicationThephospho-PRAS40(Thr246)colorimetricSTARELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtoquantifyspecificlevelsofsignalingtargetsindenaturedcellextracts.
      KeyApplications
      • ELISA
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      AnalytesAvailable
      • phospho-PRAS40(Thr246)
      EntrezGeneNumber
      EntrezGeneSummaryAKT1S1isaproline-richsubstrateofAKT(MIM164730)thatbinds14-3-3protein(seeYWHAH,MIM113508)whenphosphorylated(Kovacinaetal.,2003[PubMed12524439]).[suppliedbyOMIM]
      GeneSymbol
      • AKT1S1
      • Lobe
      • MGC2865
      • PRAS40
      Modifications
      • Phosphorylation
      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:1unit/mL
        RangeofDetection:1.6to100units/mL
        SpeciesReactivity:Human
      Dimensions
      MaterialsInformation
      MaterialsInformation
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