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Millipore/17-455 | AKT1 STAR ELISA Kit/17-455/96 assays
  • Millipore/17-455 | AKT1 STAR ELISA Kit/17-455/96 assays

Millipore/17-455 | AKT1 STAR ELISA Kit/17-455/96 assays

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货号: 17-455
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    • Description
      CatalogueNumber17-455
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      DescriptionAKT1STARELISAKit
      AlternateNames
      • PKB
      BackgroundInformationTheUPSTATEcolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheAKT1plateiscoatedwithaspecificmousemonoclonalanti-AKT1captureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingAKT1antigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-AKT1antibodytodetectthecapturedAKT1ontheplatewell.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardcurve.

      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.
      MaterialsRequiredbutNotDelivered1.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
      • 1.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)of25XELISAWashBuffer
      • 5.Anti-RabbitIgGHRPconjugate:(PartNo.17-455E)Onevial(125&micor;L)of100Xanti-rabbitHRPconjugatecontainingthimerosol
      • 6.HRPDiluent:(PartNo.17-455F)Onebottle(25mL)ofHRPDiluentcontainingthimerosol
      • 7.TMBSolution:(PartNo.17-455G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse
      • 8.StopSolution:(PartNo.17-455H)Onebottle(25mL)ofstopsolution,readytouse.
      • 9.AKT1Standard:(PartNo.17-455I)FourvialsofAKT1standard,lyophilized
      • 10.PlateCovers:Twoplatecovers.
      DetectionmethodChromogenic
      StorageandShippingInformation
      StorageConditions1yearat4°Cfromdateofshipment
      Applications
      ApplicationThisAKT1STARELISAKitisusedtomeasure&quantifyAkt(PKB)levels.
      KeyApplications
      • ELISA
      BiologicalInformation
      SpeciesReactivity
      • Human
      • Mouse
      • Rat
      AnalytesAvailable
      • Akt(PKB)
      EntrezGeneNumber
      EntrezGeneSummaryTheserine-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
      UniProtNumber
      UniProtSummaryFUNCTION: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:0.3ng/mL.
        RangeofDetection:0.3to20ng/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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