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

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貨號: 17-477

品牌: Millipore

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Description
CatalogueNumber	17-477
BrandFamily	Upstate
TradeName	STAR Upstate
Description	PRAS40STARELISAKit
AlternateNames	SignalTranscductionAssayReactionPRAS40ELISA
BackgroundInformation	I.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.
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

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.
Detectionmethod	Colorimetric

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.

Detectionmethod

Colorimetric

StorageandShippingInformation
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.

StorageandShippingInformation

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.

Applications
Application	ThePRAS40colorimetricSTARELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtoquantifyspecificlevelsofsignalingtargetsindenaturedcellextracts.
KeyApplications	ELISA

BIOLOGicalInformation
SpeciesReactivity	Human
AnalytesAvailable	PRAS40
EntrezGeneNumber	NM_001098632.1 NM_001098633.1 NM_032375.3
EntrezGeneSummary	AKT1S1isaproline-richsubstrateofAKT(MIM164730)thatbinds14-3-3protein(seeYWHAH,MIM113508)whenphosphorylated(Kovacinaetal.,2003[PubMed12524439]).[suppliedbyOMIM]
GeneSymbol	AKT1S1 Lobe MGC2865 PRAS40
UniProtNumber	Q96B36
UniProtSummary	FUNCTION: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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