微孔/17-456 |磷酸化Akt（Thr308）STAR ELISA试剂盒/17-456/96分析
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¥0.00
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产品分类：
夹心法ELISA
公司分类：
Sandwich_method_ELISA
联系Q Q：
3392242852
电话号码：
4000-520-616
电子邮箱：
info@ebiomall.com
商品介绍
| Description | |
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| CatalogueNumber | 17-456 |
| BrandFamily | Upstate |
| TradeName |
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| Description | phospho-Akt(Thr308)STARELISAKit |
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| 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,GL,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 |
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| Detectionmethod | Chromogenic |
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| StorageConditions | 1yearat4°C |
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