DescriptionCatalogueNumber17-455BrandFamilyUpstateTradeNameSTARUpstateDescriptionAKT1STARELISAKitAlternateNamesPKBBackgroundInformationTheUPSTATEcolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheAKT1plateiscoatedwithaspecificmousemonoclonalanti-AKT1captureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingAKT1antigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-AKT1antibodytodetectthecapturedAKT1ontheplatewell.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardcurve.II.AktBACKGROUNDAkt(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.MaterialsRequiredbutNotDelivered1.Multi-channelorrepeatingPipettes2.Plateshaker(optional)3.Pipettors&tipscapableofaccuratelymeasuring1-1000%micro;L4.GraduatedSEROlogicalpipettes5.96-wellmicrotiterPlateReaderwith450nmfilter6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata7.Microfugetubesforstandardandsampledilutions8.Mechanicalvortex9.1litercontainer10.DistilledordeionizedwaterProductInformationComponents1.CapturePlatepre-coatedwithanti-AKT1antibody:(PartNo.17-455A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.2.Anti-AKT1detectionantibody:(PartNo.17-455B)Onebottle(11mL)ofanti-AKT1detectionantibodycontainingsodiumazide,readytouse.3.ELISADiluent:(PartNo.17-455C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.4.25XELISAWashBuffer:(PartNo.17-455D)Onebottle(50mL)of25XELISAWashBuffer5.Anti-RabbitIgGHRPconjugate:(PartNo.17-455E)Onevial(125&micor;L)of100Xanti-rabbitHRPconjugatecontainingthimerosol6.HRPDiluent:(PartNo.17-455F)Onebottle(25mL)ofHRPDiluentcontainingthimerosol7.TMBSolution:(PartNo.17-455G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse8.StopSolution:(PartNo.17-455H)Onebottle(25mL)ofstopsolution,readytouse.9.AKT1Standard:(PartNo.17-455I)FourvialsofAKT1standard,lyophilized10.PlateCovers:Twoplatecovers.DetectionmethodChromogenicStorageandShippingInformationStorageConditions1yearat4°CfromdateofshipmentApplicationsApplicationThisAKT1STARELISAKitisusedtomeasure&quantifyAkt(PKB)levels.KeyApplicationsELISABiologicalInformationSpeciesReactivityHumanMouseRatAnalytesAvailableAkt(PKB)EntrezGeneNumberNM_001014431.1NM_001014432.1NM_005163.2EntrezGeneSummaryTheserine-threonineproteinkinaseencodedbytheAKT1geneiscatalyticallyinactiveinserum-starvedprimaryandimmortalizedfibroblasts.AKT1andtherelatedAKT2areactivatedbyplatelet-derivedgrowthfactor.Theactivationisrapidandspecific,anditisabrogatedbymutationsinthepleckstrinhomologydomainofAKT1.Itwasshownthattheactivationoccursthroughphosphatidylinositol3-kinase.InthedevelopingnervoussystemAKTisacriticalmediatorofgrowthfactor-inducedneuronalsurvival.Survivalfactorscansuppressapoptosisinatranscription-independentmannerbyactivatingtheserine/threoninekinaseAKT1,whichthenphosphorylatesandinactivatescomponentsoftheapoptoticmachinery.Multiplealternativelysplicedtranscriptvariantshavebeenfoundforthisgene.GeneSymbolAKT1RACPRKBAMGC99656RAC-ALPHARAC-PK-alphaC-AKTPKBAKTUniProtNumberP31749UniProtSummaryFUNCTION: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;55686DaSUBUNIT: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.PhysicochemicalInformationSensitivitySensitivity:0.3ng/mL.RangeofDetection:0.3to20ng/mLDimensionsMaterialsInformationMaterialsInformation