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

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貨號: 17-486

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Description
CatalogueNumber	17-486
BrandFamily	Upstate
TradeName	STAR Upstate
Description	phospho-IκB(Ser32)STARELISAKit
BackgroundInformation	I.TESTPRINCIPLE: ThecolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheI_κBplateiscoatedwithaspecificmousemonoclonalI_κBcaptureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingI_κBantigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanyun-boundnonspecificmaterial.Aspecificrabbitanti-phospho-I_κBantibodyisaddedtodetectthecapturedI_κBontheplatewellthatisphosphorylatedonSer32.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader. Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.ThekitalsoincludesastandardthatisrunasbothapositivecontrolandtogenerateastandardcurveforI_κBmeasurement. II.BACKGROUND: NF_κBsignalingisacriticalregulatornotonlyofimmunefunction,butalsoofproliferationversusapoptosisinresponsetovariousstimuli.Inamajorityofunstimulatedcells,theNF_κBtranscriptionfactorsexistintheirinactiveformandareretainedinthecytoplasmbytheboundinhibitoryI_κBproteinsthatpreventitfromenteringthenucleus,thuskeepingNF_κBinaninactivestate.Uponstimulationbymultipleinducersincludingvirusesorcytokines,suchasTNFα,IL-1,orPMA,I_κBαisrapidlyphosphorylatedbyIKK(I_κBKinase),whichphosphorylatesI_κBonserines32and36,allowingrecognitionbytheubiquitinligasemachinery,leADIngtoitspolyubiquinationanddegradation,resultinginthereleaseoftheNF_κBcomplex.OnceI_κBisdegraded,NF_κBisabletoinitiatetranscription.TheNF_κBcomplexthentranslocatestothenucleuswhereitactivatesgenetranscription.NF_κBinducesthetranscriptionofitsowninhibitor,I_κB_α,causinganautoregulatorymechanismofNF_κBactivityandgeneratingtheinactiveformofNF_κB.ThenewlyformednuclearNF_κB-I_κB_αcomplexesarethenexportedouttothecytoplasm,therebyreestablishingthecytoplasmicpoolofinactiveNF_κBcomplexesprimedforanotherroundofactivationtotakeplace.ThewidevarietyofgenesregulatedbyNF_κBincludesthoseencodingcytokines,chemokines,adhesionmolecules,acutephaseproteins,andinducIBLeeffectorenzymes.MutantI_κBinwhichserines32and36arechangedtoalanines,isnotphosphorylated,andthereforenotdegraded.CellsexpressingthisproteinarenotabletoactivateNF_κB,providingausefultooltostudytheroleofNF_κBinvariouspathwaysandprocesses.
MaterialsRequiredbutNotDelivered	1.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-I_κBantibody:(PartNo.17-486A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch. 2.Anti-phospho-I_κB(ser32)detectionantibody:(PartNo.17-486B)Onebottle(6mL)ofanti-phospho-I_κB(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.PhosphorylatedI_κB(Ser32)Standard:(PartNo.17-486I)TwovialsofphosphorylatedI_κBstandard(Ser32),lyophilized. 10.PlateCovers:Twoplatecovers.
Detectionmethod	Colorimetric

ProductInformation

Components

1.CapturePlatepre-coatedwithanti-I_κBantibody:(PartNo.17-486A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
2.Anti-phospho-I_κB(ser32)detectionantibody:(PartNo.17-486B)Onebottle(6mL)ofanti-phospho-I_κB(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.PhosphorylatedI_κB(Ser32)Standard:(PartNo.17-486I)TwovialsofphosphorylatedI_κBstandard(Ser32),lyophilized.
10.PlateCovers:Twoplatecovers.

Detectionmethod

Colorimetric

StorageandShippingInformation
StorageConditions	1yearat4°Cfromdateofshipment

Applications
Application	Thisphospho-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	NM_020529.1
EntrezGeneSummary	NFKB1(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	P25963
UniProtSummary	FUNCTION: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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