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Millipore/17-457 | phospho-Akt (Ser473) STAR ELISA Kit/17-457/96 assays

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貨號: 17-457

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Description
CatalogueNumber	17-457
BrandFamily	Upstate
TradeName	STAR Upstate
Description	phospho-Akt(Ser473)STARELISAKit
AlternateNames	PKB
BackgroundInformation	TheUPSTATEcolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheAktplateiscoatedwithaspecificmousemonoclonalAktcaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingAktantigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-phospho-Akt(Ser473)antibodytodetectthecapturedAktontheplatewellthatisphosphorylatedonSer473.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardlineofdetection. II.AktBACKGROUND Akt(ProteinKinaseB),aSer/Thrkinase,isamajorknowneffecterofthePI3KinasepathwayandisinvolvedinmultiplesignalingpathwaysthatrelatetomanyBIOLOGicalprocessesincludingglucosemetabolism,cellsurvival/apoptosis,cellcyclecontrol,angiogenesis,differentiation,andcellgrowthandproliferation.Aktisactivatedbyligand-stimulatedgrowthfactorreceptorsignalingthatactivatesthePhosphatidylinositol3-kinase(PI3Kinase,PI3K)dependentmanner.PKBisoneofthemostfrequentlyhyperactivatedproteinkinasesinhumancancers.InmammalsthreeisoformsofAkt(Akt1/PKBα,Akt2/PKBβ,andAkt3/PKBγ)exists.Theyexhibitahighdegreeofhomology,butdifferslightlyinthelocalizationoftheirregulatoryphosphorylationsites.Akt1isthepredominantisoformthatisinmosttissuesandisthoughttohaveadominantroleingrowth,survival,embryonicdevelopment,andpost-natalsurvival.Additionally,Akt1/PKBαisrequiredforADIpocytedifferentiation,whereasAkt2/PKBβandAkt3/PKBγarenot.Akt2isstronglycorrelatedwiththeregulationofglucosehomeostasisandisthepredominantPKBisoformexpressedininsulin-responsivetissueswheredefectiveAkt2resultsinimpairedinsulin-stimulatedglucoseuptakeinmuscleandadipocytes.Akt3isabundantinbraintissue.EachAktisoformiscomposedofthreefunctionallydistinctregions:anN-terminalPleckstrinHomology(PH)domainthatprovidesalipid-bindingmoduletodirectAkttoPIP3atthecellmembraneasaresultofPI3Kinase(PI3K)activitythatisnecessaryforitsactivation,acentralcatalyticdomain,andaC-terminalhydrophobicmotif.TheactivationandregulationofAKTisdependentonadualregulatorymechanismthatrequiresbothitstranslocationtotheplasmamembraneanddualphosphorylationonThr308andSer473byPDK1andtheTORC2complex,respectively.Thisisaccomplishedbythegenerationandbuild-upofPIP3byPI3KinconjunctionwithreducedPTENfunctionthatresultsintheactivationofPDK1(3-phosphoinositide-dependentproteinkinase-1)andtherecruitmentofAKTtotheplasmamembranebydirectinteractionwithitsPHdomain.TheactivatedPDK1theninturnphosphorylatesAktonThr308initsactivationloop.ThisphosphorylationisnecessaryandsufficientforAKTactivation;howevermaximalactivationrequirestheadditionalphosphorylationatSer473.Anotherkinasecomplex,recentlyidentifiedasTORC2,whichiscomposedofthemTOR,Rictor,G?L,Sin1,andProtor1and2(previouslyreferredtoastheunidentifiedkinasePDK2),phosphorylatesAKTonSer473initshydrophobicmotif.AfterAktisactivated,itisliberatedfromtheplasmamembraneandreleasedintothecytosolandnucleuswhereitinteractswithandphosphorylatesmultiplebindingpartners.Ithasbeenshowntophosphorylateover40substrates,someofwhichareactivatedbyphosphorylationsuchasmTOR,AS160,PRAS40(Thr246),IKK,MDM2,NFκB,andTSC1&2andsomethatareinhibitedbyitsphosphorylationthatincludeBad(Ser136),GSK3(Ser9),FKHR(Ser256),andCaspase9(Ser196). JustasthesetwoAKTphosphorylationsitesarephosphorylatedbytwoseparatemechanisms,theyarebothregulatedbytwodifferentphosphatases.ThedephosphorylationandsubsequentinactivationofAKTismuchlessunderstoodthanitsactivation.Itwasnotuntiltherecentdiscoveryoftwonewphosphatases,PHLPP1andPHLPP2(PHdomainleucine-richrepeatproteinphosphatase)thattheprocesswasbetterelucidated.DephosphorylationofAKTatSer473,butnotatThr308,wasfoundtobemediatedbyoneorbothofthePHLPPfamilyofphosphatases.Anothermorepromiscuousphosphatase,PP2A,isnowbelievedtodephosphorylateAKTonthePDK1phosphorylationsiteatThr308.TogetherthesephosphataseshelpregulatetheactivityofAKT.WithAKThavingsomanysignalingpartnersanditsinvolvementinmultiplesignalingpathwaysandcellularmechanisms,itisnowonderwhyAKTissowellstudiedandahighlysoughtafterdrugtarget.
MaterialsRequiredbutNotDelivered	1.Multi-channelorrepeatingPipettes 2.Plateshaker(optional) 3.Pipettors&tipscapableofaccuratelymeasuring1-1000%micro;L 4.GraduatedSEROlogicalpipettes 5.96-wellmicrotiterPlateReaderwith450nmfilter 6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata 7.Microfugetubesforstandardandsampledilutions 8.Mechanicalvortex 9.1litercontainer 10.Distilledordeionizedwater

ProductInformation
Components	CapturePlatepre-coatedwithanti-Aktantibody:Onepre-coated96-stripwellimmunoplatesealedinafoilpouch. Anti-phospho-Akt(Ser473)detectionantibody:Onebottle(6mL)ofanti-phospho-Akt(Ser473)detectionantibodycontainingsodiumazide,readytouse. ELISADiluent:Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse. 25XELISAWashBuffer:Onebottle(50mL)of25XELISAWashBuffer. Anti-RabbitIgGHRPconjugate:Onevial(125μL)of100Xanti-rabbitHRPconjugatecontainingthimerosal. HRPDiluent:Onebottle(25mL)ofHRPDiluentcontainingthimerosal. TMBSolution:Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse. StopSolution:Onebottle(25mL)ofstopsolution,readytouse. Phospho-Akt(Ser473)Standard:FourvialsofphosphorylatedAKT(Ser473)standard,lyophilized. PlateCovers:Twoplatecovers.
Detectionmethod	Chromogenic

ProductInformation

Components

CapturePlatepre-coatedwithanti-Aktantibody:Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
Anti-phospho-Akt(Ser473)detectionantibody:Onebottle(6mL)ofanti-phospho-Akt(Ser473)detectionantibodycontainingsodiumazide,readytouse.
ELISADiluent:Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
25XELISAWashBuffer:Onebottle(50mL)of25XELISAWashBuffer.
Anti-RabbitIgGHRPconjugate:Onevial(125μL)of100Xanti-rabbitHRPconjugatecontainingthimerosal.
HRPDiluent:Onebottle(25mL)ofHRPDiluentcontainingthimerosal.
TMBSolution:Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
StopSolution:Onebottle(25mL)ofstopsolution,readytouse.
Phospho-Akt(Ser473)Standard:FourvialsofphosphorylatedAKT(Ser473)standard,lyophilized.
PlateCovers:Twoplatecovers.

Detectionmethod

Chromogenic

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	Thephospho-Akt(Ser473)colorimetricSTARELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtoquantifyspecificlevelsofsignalingtargetsindenaturedcellextracts.
KeyApplications	ELISA

BiologicalInformation
SpeciesReactivity	Human Mouse Rat
AnalytesAvailable	Akt(PKB)
EntrezGeneNumber	NM_001014431.1 NM_001014432.1 NM_005163.2
EntrezGeneSummary	Theserine-threonineproteinkinaseencodedbytheAKT1geneiscatalyticallyinactiveinserum-starvedprimaryandimmortalizedfibroblasts.AKT1andtherelatedAKT2areactivatedbyplatelet-derivedgrowthfactor.Theactivationisrapidandspecific,anditisabrogatedbymutationsinthepleckstrinhomologydomainofAKT1.Itwasshownthattheactivationoccursthroughphosphatidylinositol3-kinase.InthedevelopingnervoussystemAKTisacriticalmediatorofgrowthfactor-inducedneuronalsurvival.Survivalfactorscansuppressapoptosisinatranscription-independentmannerbyactivatingtheserine/threoninekinaseAKT1,whichthenphosphorylatesandinactivatescomponentsoftheapoptoticmachinery.Multiplealternativelysplicedtranscriptvariantshavebeenfoundforthisgene.
GeneSymbol	AKT1 RAC PRKBA MGC99656 RAC-ALPHA RAC-PK-alpha C-AKT PKB AKT
Modifications	Phosphorylation
UniProtNumber	P31749
UniProtSummary	FUNCTION:SwissProt:P31749#Generalproteinkinasecapableofphosphorylatingseveralknownproteins.PhosphorylatesTBC1D4.Signalsdownstreamofphosphatidylinositol3-kinase(PI(3)K)tomediatetheeffectsofvariousgrowthfactorssuchasplatelet-derivedgrowthfactor(PDGF),epidermalgrowthfactor(EGF),insulinandinsulin-likegrowthfactorI(IGF-I).Playsaroleinglucosetransportbymediatinginsulin-inducedtranslocationoftheGLUT4glucosetransportertothecellsurface.MediatestheantiapoptoticeffectsofIGF-I.Mediatesinsulin-stimulatedproteinsynthesis,partlybyplayingaroleinbothinsulin-inducedphosphorylationof4E-BP1andininsulin-inducedactivationofp70S6kinase.Promotesglycogensynthesisbymediatingtheinsulin-inducedactivationofglycogensynthase. SIZE:480aminoacids;55686Da SUBUNIT:InteractswithCENTG1isoform2(PIKE-A)inthepresenceofguaninenucleotides.TheC-terminusinteractswithCCDC88A/GRDN.InteractswithAKTIP. SUBCELLULARLOCATION:Cytoplasm.Nucleus.Note=Nucleusafteractivationbyintegrin-linkedproteinkinase1(ILK1). TISSUESPECIFICITY:Inallhumancelltypessofaranalyzed. DOMAIN:SwissProt:P31749BindingofthePHdomaintothephosphatidylinositol3-kinasealpha(PI(3)K)resultsinitstargetingtotheplasmamembrane. PTM:PhosphorylationonThr-308,Ser-473andTyr-474isrequiredforfullactivity.Ser-473phosphorylationbytheRictor-mTorcomplexfavorsThr-308phosphorylationbyPDPK1.Ser-473phosphorylationisenhancedbyinteractionwithCENTG1isoform2(PIKE-A). SIMILARITY:Belongstotheproteinkinasesuperfamily.AGCSer/Thrproteinkinasefamily.RACsubfamily.&Contains1AGC-kinaseC-terminaldomain.&Contains1PHdomain.&Contains1proteinkinasedomain.

PhysicochemicalInformation
Sensitivity	Sensitivity:0.3units/mLRangeofDetection:0.4to25units/mL

Dimensions

MaterialsInformation

MaterialsInformation

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Millipore/17-457 | phospho-Akt (Ser473) STAR ELISA Kit/17-457/96 assays

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