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

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貨號: 17-456

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Description
CatalogueNumber	17-456
BrandFamily	Upstate
TradeName	STAR Upstate
Description	phospho-Akt(Thr308)STARELISAKit
AlternateNames	PKB
BackgroundInformation	TheUPSTATEcolorimetricSTAR(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.
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	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.
Detectionmethod	Chromogenic

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.

Detectionmethod

Chromogenic

StorageandShippingInformation
StorageConditions	1yearat4°C

Applications
Application	Thephospho-Akt(Thr308)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

PhysicochemicalInformation
Sensitivity	Sensitivity:1unit/mL. RangeofDetection:1.6to100units/mL

Dimensions

MaterialsInformation

MaterialsInformation

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

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