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Millipore/17-456 | phospho-Akt (Thr308) STAR ELISA Kit/17-456/96 assays
  • Millipore/17-456 | phospho-Akt (Thr308) STAR ELISA Kit/17-456/96 assays

Millipore/17-456 | phospho-Akt (Thr308) STAR ELISA Kit/17-456/96 assays

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货号: 17-456
品牌: Millipore
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    • Description
      CatalogueNumber17-456
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      Descriptionphospho-Akt(Thr308)STARELISAKit
      AlternateNames
      • PKB
      BackgroundInformationTheUPSTATEcolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheAKTplateiscoatedwithaspecificmousemonoclonalanti-AKTcaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingAKTantigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-AKTantibodytodetectthecapturedphospho-AKT(Thr308)ontheplatewell.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-AKTantibody:(PartNo.17-456A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • 2.Anti-phospho-Akt(Thr308)detectionantibody:(PartNo.17-456B)Onebottle(11mL)ofanti-phospho-Akt(Thr308)detectionantibodycontainingsodiumazide,readytouse.
      • 3.ELISADiluent:(PartNo.17-456C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 4.25XELISAWashBuffer:(PartNo.17-456D)Onebottle(50mL)of25XELISAWashBuffer.
      • 5.Anti-RabbitIgGHRPconjugate:(PartNo.17-456E)Onevial(125µL)of100Xanti-rabbitHRPconjugatecontainingthimerosol.
      • 6.HRPDiluent:(PartNo.17-456F)Onebottle(25mL)ofHRPDiluentcontainingthimerosol.
      • 7.TMBSolution:(PartNo.17-456G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • 8.StopSolution:(PartNo.17-456H)Onebottle(25mL)ofstopsolution,readytouse.
      • 9.Phospho-Akt(Thr308)Standard:(PartNo.17-456I)Fourvialsofphospho-Akt(Thr308)standard,lyophilized.
      • 10.PlateCovers:Twoplatecovers.
      DetectionmethodChromogenic
      StorageandShippingInformation
      StorageConditions1yearat4°C
      Applications
      ApplicationThephospho-Akt(Thr308)colorimetricSTARELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtoquantifyspecificlevelsofsignalingtargetsindenaturedcellextracts.
      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
      Modifications
      • Phosphorylation
      UniProtNumber
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:1unit/mL.
        RangeofDetection:1.6to100units/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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