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Millipore/17-485 | IκB STAR ELISA Kit/17-485/96 assays
  • Millipore/17-485 | IκB STAR ELISA Kit/17-485/96 assays

Millipore/17-485 | IκB STAR ELISA Kit/17-485/96 assays

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货号: 17-485
品牌: Millipore
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    • Description
      CatalogueNumber17-485
      TradeName
      • STAR
      • Upstate
      DescriptionIκBSTARELISAKit
      BackgroundInformationTestPrinciple
      TheUPSTATE®colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheIkBplateiscoatedwithaspecificmousemonoclonalIkBcaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingIkBantigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-IkBantibodytodetectthecaptureIkBontheplatewell.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.
      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardlineofdetection.


      IkBBackground
      IKKbeta(IkBKinase,IKKb,I-Kappa-Bkinase-beta)isamemberoftheIKKcomplexwhichiscomposedofIKKalpha,IKKbeta,IKKgammaandIKAP.IkBKinase(IKK)complexisacriticalcomponentinNFBregulation.Inamajorityofunstimulatedcells,theNFκBtranscriptionfactorsexistintheirinactiveformandareretainedinthecytoplasmbytheboundinhibitoryIkBproteins.ForNFBtobecomeactivated,itmustfirstdisassociatefromtheinhibitorIκB.ThisoccursviatheserinephosphorylationofIkBonSer32andSer36.Postreceptorstimulation,mostnotablybyTNFα,NIK,aserine/threoninekinase,becomesactivatesandphosphorylatesIKKαandIKKβ.Phosphorylationof2sitesattheactivationloopofIKKbetaisessentialforactivationofIKKbyTNFandIL1.Onceactivated,IKKbetaautophosphorylates,whichinturndecreasesIKKactivityandpreventsprolongedactivationoftheinflammatoryresponse.Additionally,IKKbetaactivitycanalsoberegulatedbyMEKK-1.IKKthenbindstoandphosphorylatesIkBαonthetwoserineresidues.ThisphosphorylationmarksIkBforubiquinationanddestructionbytheproteosome.ThisfreetheNFkBcomplexandallowsittotranslocatestothenucleus.
      MaterialsRequiredbutNotDelivered1.Multi-channelorrepeatingPipettes
      2.Plateshaker(optional)
      3.Pipettors&tipscapableofaccuratelymeasuring1-1000μL
      4.GraduatedSEROlogicalpipettes
      5.96-wellmicrotiterPlateReaderwith450nmfilter
      6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata
      7.Microfugetubesforstandardandsampledilutions
      8.Mechanicalvortex
      9.1litercontainer
      10.Distilledordeionizedwater
      ProductInformation
      Components
      • 1.CapturePlatepre-coatedwithanti-IκBantibody:(PartNo.17-485A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • 2.Anti-IκBdetectionantibody:(PartNo.17-485B)Onebottle(11mL)ofanti-IκBdetectionantibodycontainingsodiumazide,readytouse.
      • 3.ELISADiluent:(PartNo.17-485C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 4.25XELISAWashBuffer:(PartNo.17-485D)Onebottle(50mL)of25XELISAWashBuffer.
      • 5.Anti-RabbitIgGHRPconjugate:(PartNo.17-485E)Onevial(125μL)of100Xanti-rabbitHRPconjugate.
      • 6.HRPDiluent:(PartNo.17-485F)Onebottle(25mL)ofHRPDiluent.
      • 7.TMBSolution:(PartNo.17-485G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • 8.StopSolution:(PartNo.17-485H)Onebottle(25mL)ofstopsolution,readytouse.
      • 9.IκBStandard:(PartNo.17-485I)TwovialsofIκBstandard,lyophilized.
      • 10.PlateCovers:Twoplatecovers.
      DetectionmethodColorimetric
      StorageandShippingInformation
      StorageConditions1yearat4°Cfromdateofshipment
      Applications
      ApplicationTheIκBcolorimetricSTARELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtoquantifyspecificlevelsofsignalingtargetsindenaturedcellextracts.
      KeyApplications
      • ELISA
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      AnalytesAvailable
      • IκB
      EntrezGeneNumber
      EntrezGeneSummaryNFKB1(MIM164011)orNFKB2(MIM164012)isboundtoREL(MIM164910),RELA(MIM164014),orRELB(MIM604758)toformtheNFKBcomplex.TheNFKBcomplexisinhibitedbyI-kappa-Bproteins(NFKBIAorNFKBIB,MIM604495),whichinactivateNF-kappa-Bbytrappingitinthecytoplasm.PhosphorylationofserineresiduesontheI-kappa-Bproteinsbykinases(IKBKA,MIM600664,orIKBKB,MIM603258)marksthemfordestructionviatheubiquitinationpathway,therebyallowingactivationoftheNF-kappa-Bcomplex.ActivatedNFKBcomplextranslocatesintothenucleusandbindsDNAatkappa-B-bindingmotifssuchas5-primeGGGRNNYYCC3-primeor5-primeHGGARNYYCC3-prime(whereHisA,C,orT;RisanAorGpurine;andYisaCorTpyrimidine).[suppliedbyOMIM]
      GeneSymbol
      • NFKBIA
      • IKB-alpha
      • IKBA
      • I-kappa-B-alpha
      • MAD-3
      • MAD3
      • IkappaBalpha
      • NFKBI
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:P25963#InhibitsNF-kappa-Bbycomplexingwithandtrappingitinthecytoplasm.MaybeinvolvedinregulationoftranscriptionalresponsestoNF-kappa-B,includingcelladhesion,immuneandproinflammatoryresponses,apoptosis,differentiationandgrowth.Controlledbysequentialserine-phosphorylation,ubiquitinationanddegradation.Tyrosine-phosphorylationcouldonlyleadtodissociationfromNF-kappa-B.
      SIZE:317aminoacids;35609Da
      SUBUNIT:Interactswithp65(RELA).InteractswithNKIRAS1andNKIRAS2.InteractswithHBVproteinX.
      SUBCELLULARLOCATION:Cytoplasm.
      PTM:Phosphorylated;disablesinhibitionofNF-kappa-BDNA-bindingactivity.&Ubiquitinated;subsequenttostimulus-dependentphosphorylationonserines,polyubiquitinationtargetstheproteinforrapiddegradationviatheubiquitinsystem.
      DISEASE:SwissProt:P25963#DefectsinNFKBIAareacauseofautosomaldominantanhidroticectodermaldysplasiawithimmunodeficiency(AD-EDA-ID)[MIM:164008].Ectodermaldysplasias(EDs)constituteaheterogeneousgroupofdevelopmentaldisordersaffectingtissuesofectodermalorigin.EDsarecharacterizedbyabnormaldevelopmentoftwoormoreectodermalstructuressuchashair,teeth,nailsandsweatglands,withorwithoutanyadditionalclinicalsign.Eachcombinationofclinicalfeaturesrepresentsadifferenttypeofectodermaldysplasia.
      SIMILARITY:SwissProt:P25963##BelongstotheNF-kappa-Binhibitorfamily.&Contains5ANKrepeats.
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:62.5pg/mL.
        RangeofDetection:62.5to4000pg/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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