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Millipore/17-486 | phospho-IκB (Ser32) STAR ELISA Kit/17-486/96 assays
  • Millipore/17-486 | phospho-IκB (Ser32) STAR ELISA Kit/17-486/96 assays

Millipore/17-486 | phospho-IκB (Ser32) STAR ELISA Kit/17-486/96 assays

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货号: 17-486
品牌: Millipore
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    • Description
      CatalogueNumber17-486
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      Descriptionphospho-IκB(Ser32)STARELISAKit
      BackgroundInformationI.TESTPRINCIPLE:
      ThecolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheIκBplateiscoatedwithaspecificmousemonoclonalIκBcaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingIκBantigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanyun-boundnonspecificmaterial.Aspecificrabbitanti-phospho-IκBantibodyisaddedtodetectthecapturedIκBontheplatewellthatisphosphorylatedonSer32.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.
      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.ThekitalsoincludesastandardthatisrunasbothapositivecontrolandtogenerateastandardcurveforIκBmeasurement.

      II.BACKGROUND:
      NFκBsignalingisacriticalregulatornotonlyofimmunefunction,butalsoofproliferationversusapoptosisinresponsetovariousstimuli.Inamajorityofunstimulatedcells,theNFκBtranscriptionfactorsexistintheirinactiveformandareretainedinthecytoplasmbytheboundinhibitoryIκBproteinsthatpreventitfromenteringthenucleus,thuskeepingNFκBinaninactivestate.Uponstimulationbymultipleinducersincludingvirusesorcytokines,suchasTNFα,IL-1,orPMA,IκBαisrapidlyphosphorylatedbyIKK(IκBKinase),whichphosphorylatesIκBonserines32and36,allowingrecognitionbytheubiquitinligasemachinery,leADIngtoitspolyubiquinationanddegradation,resultinginthereleaseoftheNFκBcomplex.OnceIκBisdegraded,NFκBisabletoinitiatetranscription.TheNFκBcomplexthentranslocatestothenucleuswhereitactivatesgenetranscription.NFκBinducesthetranscriptionofitsowninhibitor,IκBα,causinganautoregulatorymechanismofNFκBactivityandgeneratingtheinactiveformofNFκB.ThenewlyformednuclearNFκB-IκBαcomplexesarethenexportedouttothecytoplasm,therebyreestablishingthecytoplasmicpoolofinactiveNFκBcomplexesprimedforanotherroundofactivationtotakeplace.ThewidevarietyofgenesregulatedbyNFκBincludesthoseencodingcytokines,chemokines,adhesionmolecules,acutephaseproteins,andinducIBLeeffectorenzymes.MutantIκBinwhichserines32and36arechangedtoalanines,isnotphosphorylated,andthereforenotdegraded.CellsexpressingthisproteinarenotabletoactivateNFκB,providingausefultooltostudytheroleofNFκBinvariouspathwaysandprocesses.
      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.17-486A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • 2.Anti-phospho-IκB(ser32)detectionantibody:(PartNo.17-486B)Onebottle(6mL)ofanti-phospho-IκB(Ser32)detectionantibodycontainingsodiumazide,readytouse.
      • 3.ELISADiluent:(PartNo.17-486C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 4.25XELISAWashBuffer:(PartNo.17-486D)Onebottle(50mL)of25XELISAWashBuffer.
      • 5.Anti-RabbitIgGHRPconjugate:(PartNo.17-486E)Onevial(125μL)of100Xanti-rabbitHRPconjugate.
      • 6.HRPDiluent:(PartNo.17-486F)Onebottle(25mL)ofHRPDiluent.
      • 7.TMBSolution:(PartNo.17-486G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • 8.StopSolution:(PartNo.17-486H)Onebottle(25mL)ofstopsolution,readytouse.
      • 9.PhosphorylatedIκB(Ser32)Standard:(PartNo.17-486I)TwovialsofphosphorylatedIκBstandard(Ser32),lyophilized.
      • 10.PlateCovers:Twoplatecovers.
      DetectionmethodColorimetric
      StorageandShippingInformation
      StorageConditions1yearat4°Cfromdateofshipment
      Applications
      ApplicationThisphospho-IκB(Ser32)STARELISAKitisusedtomeasure&quantifyIκBlevels.
      KeyApplications
      • ELISA
      ApplicationNotes•Allkitreagentsshouldbebroughttoroomtemperature(20°Cto25°C)priortouse.
      •Donotusereagentsbeyondtheexpirationdateofthekit.
      •Donotmixorinterchangereagentfromvariouskitlots.
      •ManualPlateWashing:Vigorouswashingandcompleteremovalofallliquidbyaspirationattheendofeachwashingstepisveryimportanttoobtainlowbackgroundvalues.Gentleagitationduringthewashstepsora2-3minutesoakmayreducebackgroundvalues.
      •Thedesiccantenclosedinthe96-wellcaptureplatepouchwillkeeptheplatestablewhenstoredat2°to8°Cshouldthepouchlooseitssealduringshipping.
      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
      Modifications
      • Phosphorylation
      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:1Unit/mL.
        RangeofDetection:1.6to100Units/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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