Description
CatalogueNumber	17-455
BrandFamily	Upstate
TradeName	STAR Upstate
Description	AKT1STARELISAKit
AlternateNames	PKB
BackgroundInformation	TheUPSTATEcolorimetricSTAR(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.
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-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.
Detectionmethod	Chromogenic

StorageandShippingInformation
StorageConditions	1yearat4°Cfromdateofshipment

Applications
Application	ThisAKT1STARELISAKitisusedtomeasure&quantifyAkt(PKB)levels.
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
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:0.3ng/mL. RangeofDetection:0.3to20ng/mL

Dimensions

MaterialsInformation

MaterialsInformation

Millipore Immobilon -P转印膜   PVDF膜 ·适用于免疫印迹、斑点印迹和其他蛋白质印迹的最佳PVDF膜；·Western Blotting、吸附分析、氨基酸分析、N端蛋白质测序、Dot/Slot blotting、糖蛋白显色和脂多糖分析；·与他相容的染液/染色剂：考马斯亮蓝染色剂、酰胺黑、印度墨水、丽春红、胶体金、CPTS、甲苯胺蓝、透射显影和Sypro Ruby染料. 产品名称Immobilon-P说    明应用于>20KD蛋白质的Western, dot/slot blotting和其他印迹孔    径0.45um尺寸cm×cm26.5×375数    量1卷目录编号IPVH00010 ――――――――――――――――――――――――――――――――――――――― Immobilon - NC膜，混合纤维素酯（Millipore） Immobilon-NC HATF由混合纤维素酯构成，不含细胞生长期间影响细胞完整的表面活性剂。使用这些膜可从事菌落转移实验。Immobilon-NC HAHY由混合纤维素酯构成，但含有转印过程期间能改善可湿性便于操作的表面活性剂。膜的孔径是0.45μm。用于Southern blotting，Northern Blotting，Western blotting，Colony/plaque Lifts，Dot/slot blots，吸附分析，细胞培养.。 产品名称Immobilon - NC说    明应用于Southern杂交， Northern杂交，免疫印迹，结合实验孔    径0.45um尺寸cm×cm33×300数    量1卷目录编号HATF00010