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Millipore/17-475 | Phospho-p53 (Ser15) STAR ELISA Kit/17-475/96 assays
  • Millipore/17-475 | Phospho-p53 (Ser15) STAR ELISA Kit/17-475/96 assays

Millipore/17-475 | Phospho-p53 (Ser15) STAR ELISA Kit/17-475/96 assays

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貨號: 17-475
品牌: Millipore
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    • Description
      CatalogueNumber17-475
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      DescriptionPhospho-p53(Ser15)STARELISAKit
      BackgroundInformationI.TESTPRINCIPLE
      ThecolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.Thep53plateiscoatedwithaspecificmousemonoclonalp53captureantibodyonthemicrowellsofthe96-wellclearplate.Samplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingp53antigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanyunboundnon-specificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-phospho-p53(Ser15)antibodytodetectthecapturedp53ontheplatewellthatisphosphorylatedonSer15.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Thisallowsforasensitiveenzymaticdetectionofthesample.

      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtogenerateastandardcurveforphosphorylatedp53(Ser15)measurement.

      II.p53BACKGROUND
      p53,agenethatismutatedinapproximatelyhalfofallhumancancers,haslongbeenregardedastheguardianofthegenomebyregulatinggenescontrollingcellcycleprogression,DNArepairandapoptosis.Inresponsetocellularstress,suchasDNAdamage,hypoxia,orrADIation,p53isactivatedandinturnittranscriptionallyactivatestheexpressionofparticulargenes,includingthecyclin-dependentkinaseinhibitorp21,andtogetherwithp19ARF,inducesexpressionofp21Cip1,tocausecellcyclearrest.Alternatively,p53canworkviaapoptosisasawayofeliminatingirreparablydamagedcells.

      Inactivationorlossofp53hasbeenassociatedwithderegulationofthecellcycleandDNAreplication,inefficientDNArepair,lossofcellularapoptoticresponsesandultimatelythedevelopmentofvarioushumancancers.Thep53polypeptidecontainsthreedistinctregions.Theamino-terminal83aminoacidsofp53containsthetransactivationdomain,aswellastheregioninvolvedintranscription-independentgrowthsuppression,thecentralsequence-specificDNAbindingregion,andthecarboxy-terminalregioncontainstheDNA-bindingdomain.ActivationandstABIlizationofp53isregulatedbyphosphorylationandpossIBLyacetylation
      MaterialsRequiredbutNotDelivered1.Multi-channelorrepeatingPipettes
      2.Plateshaker(optional)
      3.Pipettorsandtipscapableofaccuratelymeasuring1-1000μL
      4.GraduatedSEROlogicalpipettes
      5.96-wellmicrotiterPlateReaderwith450nmfilter
      6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata
      7.Microfugetubesforstandardandsampledilutions
      8.Mechanicalvortex
      9.1litercontainer
      10.Distilledordeionizedwater
      ProductInformation
      Components
      • CapturePlatepre-coatedwithanti-p53antibody:(PartNo.17-475A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • Anti-phospho-p53(Ser15)detectionantibody:(PartNo.17-475B)Onebottle(11mL)ofanti-phospho-p53(Ser15)detectionantibodycontainingsodiumazide,readytouse.
      • ELISADiluent:(PartNo.17-475C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 25XELISAWashBuffer:(PartNo.17-475D)Onebottle(50mL)of25XELISAWashBuffer.
      • Anti-RabbitIgGHRPconjugate:(PartNo.17-475E)Onevial(125μL)of100Xanti-rabbitHRPconjugate
      • HRPDiluent:(PartNo.17-475F)Onebottle(25mL)ofHRPDiluent.
      • TMBSolution:(PartNo.17-475G)Onebottle(25mL)ofstabilizedtetramethylbenzidine(TMB),readytouse.
      • StopSolution:(PartNo.17-475H)Onebottle(25mL)ofstopsolution,readytouse.
      • Phospho-p53(Ser15)Standard:(PartNo.17-475I)Twovialsofphosphorylatedp53(Ser15)standard,lyophilized.
      • PlateCovers:Twoplatecovers
      DetectionmethodColorimetric
      StorageandShippingInformation
      StorageConditionsMaintaintheunopenedkitat2-8°Cuntilexpirationdate.

      Precautions
      ?Theinstructionsprovidedhavebeendesignedtooptimizethekit"sperformance.Deviationfromtheinstructionsmayresultinsuboptimalperformanceofthekitandthefailuretoproduceaccuratedata.
      ?CausticMaterial:StopSolution.Caution:Eye,hand,face,andclothingprotectionshouldbewornwhenhandlingthismaterial.
      ?SafetyWarningsandPrecautions:Thiskitisdesignedforresearchuseonlyandnotrecommendedforinternaluseinhumansoranimals.Allchemicalsshouldbeconsideredpotentiallyhazardousandprinciplesofgoodlaboratorypracticeshouldbefollowed.
      ?TheDetectionAntibodyandELISADiluentcontainsodiumazide.Sodiumazidemayreactwithcopperandleadplumbingtoformhighlyexplosivemetalazides.Upondisposal,flushwithlargeamountsofwatertopreventazidebuild-up.Avoidcontactwithskin.
      ?TheAnti-RabbitIgGHRPConjugateandHRPDiluentcontainthimerosal.Thimerosalishighlytoxicbyinhalation,contactwithskinorifswallowed.Thimerosalisapossiblemutagenandshouldbehandledaccordingly.
      Applications
      KeyApplications
      • ELISA
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      AnalytesAvailable
      • Phospho-p53(Ser15)
      EntrezGeneNumber
      EntrezGeneSummaryTumorproteinp53,anuclearprotein,playsanessentialroleintheregulationofcellcycle,specificallyinthetransitionfromG0toG1.Itisfoundinverylowlevelsinnormalcells,however,inavarietyoftransformedcelllines,itisexpressedinhighamounts,andbelievedtocontributetotransformationandmalignancy.p53isaDNA-bindingproteincontainingDNA-binding,oligomerizationandtranscriptionactivationdomains.Itispostulatedtobindasatetramertoap53-bindingsiteandactivateexpressionofdownstreamgenesthatinhibitgrowthand/orinvasion,andthusfunctionasatumorsuppressor.Mutantsofp53thatfrequentlyoccurinanumberofdifferenthumancancersfailtobindtheconsensusDNAbindingsite,andhencecausethelossoftumorsuppressoractivity.AlterationsoftheTP53geneoccurnotonlyassomaticmutationsinhumanmalignancies,butalsoasgermlinemutationsinsomecancer-pronefamilieswithLi-Fraumenisyndrome.
      GeneSymbol
      • TP53
      • P53
      • TRP53
      • p53
      • LFS1
      Modifications
      • Phosphorylation
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:P04637#Actsasatumorsuppressorinmanytumortypes;inducesgrowtharrestorapoptosisdependingonthephysiologicalcircumstancesandcelltype.Involvedincellcycleregulationasatrans-activatorthatactstonegativelyregulatecelldivisionbycontrollingasetofgenesrequiredforthisprocess.Oneoftheactivatedgenesisaninhibitorofcyclin-dependentkinases.ApoptosisinductionseemstobemediatedeitherbystimulationofBAXandFASantigenexpression,orbyrepressionofBcl-2expression.
      COFACTOR:Binds1zincionpersubunit.
      SIZE:393aminoacids;43653Da
      SUBUNIT:InteractswithAXIN1.ProbablypartofacomplexconsistingofTP53,HIPK2andAXIN1(Bysimilarity).BindsDNAasahomotetramer.InteractswithhistoneacetyltransferasesEP300andmethyltransferasesHRMT1L2andCARM1,andrecruitsthemtopromoters.Invitro,theinteractionofTP53withcancer-associated/HPV(E6)viralproteinsleadstoubiquitinationanddegradationofTP53givingapossiblemodelforcellgrowthregulation.Thiscomplexformationrequiresanadditionalfactor,E6-AP,whichstablyassociateswithTP53inthepresenceofE6.C-terminusinteractswithTAF1,whenTAF1ispartoftheTFIIDcomplex.InteractswithING4andthisinteractionmaybeindirect.FoundinacomplexwithCABLES1andTP73.InteractswithHIPK1,HIPK2,andP53DINP1.InteractswithWWOX.MayinteractswithHCVcoreprotein.InteractswithUSP7andSYVN1.InteractswithHSP90AB1(Bysimilarity).InteractswithBANP.
      SUBCELLULARLOCATION:Cytoplasm.Nucleus.Endoplasmicreticulum.Note=InteractionwithBANPpromotesnuclearlocalization.
      DOMAIN:SwissProt:P04637Thenuclearexportsignalactsasatranscriptionalrepressiondomain.
      PTM:Acetylated.AcetylationofLys-382byCREBBPenhancestranscriptionalactivity.DeacetylationofLys-382bySIRT1impairsitsabilitytoinduceproapoptoticprogramandmodulatecellsenescence.&PhosphorylationonSerresiduesmediatestranscriptionalactivation.PhosphorylatedbyHIPK1(Bysimilarity).PhosphorylatedonThr-18byVRK1,whichmaypreventtheinteractionwithMDM2.PhosphorylatedonThr-55byTAF1,whichpromotesMDM2-mediateddegradation.PhosphorylatedonSer-46byHIPK2uponUVirradiation.PhosphorylationonSer-46isrequiredforacetylationbyCREBBP.PhosphorylatedonSer-392followingUVbutnotgammairradiation.PhosphorylateduponDNAdamage,probablybyATMorATR.PhosphorylatedonSer-15uponultravioletirradiation;whichisenhancedbyinteractionwithBANP.&DephosphorylatedbyPP2A.SV40smallTantigeninhibitsthedephosphorylationbytheACformofPP2A.&MaybeO-glycosylatedintheC-terminalbasicregion.StudiedinEB-1cellline.&UbiquitinatedbySYVN1,whichleadstoproteasomaldegradation.
      DISEASE:SwissProt:P04637#TP53isfoundinincreasedamountsinawidevarietyoftransformedcells.TP53isfrequentlymutatedorinactivatedinabout60%ofcancers.&DefectsinTP53areinvolvedinesophagealsquamouscellcarcinoma(ESCC)[MIM:133239].ESCCisatumoroftheesophagus.&DefectsinTP53areacauseofLi-Fraumenisyndrome(LFS)[MIM:151623].LFSisanautosomaldominantfamilialcancersyndromethatinitsclassicformisdefinedbytheexistenceofbothaprobandwithasarcomaandtwootherfirst-degreerelativeswithacancerbyage45years.Inthesefamiliestheaffectedrelativesdevelopadiversesetofmalignanciesatunusuallyearlyages.ThespectrumofcancersinLFSincludesbreastcarcinomas,soft-tissuesarcomas,braintumors,osteosarcoma,leukemiaandadreno-corticalcarcinoma.OtherpossiblecomponenttumorsofLFSaremelanoma,gonadalcelltumorsandcarcinomasofthelung,pancreasandprostate.&DefectsinTP53maybeassociatedwithnasopharyngealcarcinoma[MIM:161550];alsoknownasnasopharyngealcancer.&DefectsinTP53arefoundinBarrettmetaplasia;alsoknownasBarrettesophagus.Itisaconditioninwhichthenormallystratifiedsquamousepitheliumoftheloweresophagusisreplacedbyametaplasticcolumnarepithelium.Theconditiondevelopsasacomplicationinapproximately10%ofpatientswithchronicgastroesophagealrefluxdiseaseandpredisposestothedevelopmentofesophagealadenocarcinoma.&DefectsinTP53areinvolvedinheadandnecksquamouscellcarcinomas(HNSCC)[MIM:275355].&DefectsinTP53areinvolvedinoralsquamouscellcarcinoma(OSCC).Cigarettesmokeisaprimemutagenicagentincanceroftheaerodigestivetract.&DefectsinTP53areacauseoflungcancer[MIM:211980].&DefectsinTP53areacauseofchoroidplexuspapilloma[MIM:260500].Choroidplexuspapillomaisaslow-growingbenigntumorofthechoroidplexusthatofteninvadestheleptomeninges.Inchildrenitisusuallyinalateralventriclebutinadultsitismoreofteninthefourthventricle.Hydrocephalusiscommon,eitherfromobstructionorfromtumorsecretionofcerebrospinalfluid.Ifitundergoesmalignanttransformationitiscalledachoroidplexuscarcinoma.Primarychoroidplexustumorsarerareandusuallyoccurinearlychildhood.&DefectsinTP53areacauseofoneformofhereditaryadrenocorticalcarcinoma(ADCC)[MIM:202300].ADCCisararechildhoodtumor,representingabout0.4%ofchildhoodtumors,withahighincidenceofassociatedtumors.ADCCoccurswithincreasedfrequencyinpatientswiththeBeckwith-Wiedemannsyndrome[MIM:130650]andisacomponenttumorinLi-Fraumenisyndrome[MIM:151623].
      SIMILARITY:Belongstothep53family.
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:1units/mL
        RangeofDetection:1.6to100units/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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