Description
CatalogueNumber	17-486
BrandFamily	Upstate
TradeName	STAR Upstate
Description	phospho-IκB(Ser32)STARELISAKit
BackgroundInformation	I.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.
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-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.
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

Millipore 提供三种不同的 Immobilon PVDF 膜，是针对不同的蛋白质印迹应用的理想选择。 现在我们还 提供采用按规格裁切的印迹方片膜和两层过滤纸组成的印迹三明治。   与硝酸纤维素膜相比，Immobilon 膜具有多种优点。 它们不易破裂或卷 曲，便于裁剪且在切割过程中不会碎裂。 它们还有低背景、广泛溶剂相容性以及优异的着色能力。 此外，它们可以重复检测。 Immobilon-P 膜（沃比森-供应） ·         0.45μm 孔径 ·         建议用于大多数免疫印迹，特别在蛋 白质大于 20kDa 时 ·         Millipore 的快速免疫检测实验方法不需要膜封闭并缩短洗涤时间，将检测时间至少减少了两个小时，而不致降低灵敏 度或特异性 用于高通量实验室的印迹三明治 ·         当您需要处理多个印迹时，印迹三明治是一种便利省时的选择。 我们的印迹三明 治包含多片 Immobilon-P 膜，其中插入了按规格裁切的多张层析级印迹纸。 转印滤纸 ·         按照大多数免疫印迹尺寸进行裁切的层析级转印滤纸。 Immobilon-PSQ 膜（沃比森-供应） ·         0.2μm 孔径和大的内表面面积 ·         较其它膜具有更高的 蛋白质吸附率和测序得率 ·         建议用于小于 20kDa 的印迹蛋白质 ·         防止小分子量的蛋白质的渗漏 Immobilon-FL 膜（沃比森-供应） ·        针对荧光应用进行优化的转印膜 ·         极低的背景提高了所 有荧光检测实验的灵敏度 ·         与所有常用的荧光探针（所有激发和发射波长）兼容 ·         是多态性检测和化学荧光应用的理想选择