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Millipore/17-479 | STAR FAK ELISA Kit/17-479/96 assays
  • Millipore/17-479 | STAR FAK ELISA Kit/17-479/96 assays

Millipore/17-479 | STAR FAK ELISA Kit/17-479/96 assays

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货号: 17-479
品牌: Millipore
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    • Description
      CatalogueNumber17-479
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      DescriptionSTARFAKELISAKit
      AlternateNames
      • Focaladhesionkinase1
      • FADK1
      • Focaladhesionkinase-relatednonkinase
      • FRNK
      • Proteinphosphatase1regulatorysubunit71
      • PPP1R71
      • Protein-tyrosinekinase2
      • p125FAK
      • pp125FAK
      BackgroundInformationI.TESTPRINCIPLE
      TheUPSTATE®colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheFAKplateiscoatedwithaspecificmousemonoclonalFAKcaptureantibody.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingFAKantigentobecaptured.Theplateisthenwashedtoremoveanyun-boundnon-specificmaterial.Aspecificrabbitanti-FAKantibodyisaddedtodetectthecapturedFAKontheplate.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Thisallowsforasensitiveenzymaticdetectionofthesample.
      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.ThekitalsoincludesastandardthatisrunasbothapositivecontrolandtogenerateastandardcurveforFAKmeasurement.

      II.FAKBACKGROUND
      FAK(FocalAdhesionKinase)isanon-receptortyrosinekinasewhichlocalizestofocaladhesionsandplaysakeyroleinintegrin-mediatedsignalingpathwaysregulatingcellmigration.Itautophosphorylatesontyrosine397duringadhesionandspreADIng,andisthoughttobeacriticaltransducerofadhesion-dependentgrowthandsurvival.FAKanditphosphorylationstateshavebeenimplicatedincancermetastasisandtumorcellsurvivalandadhesion-independentgrowth.Additionally,recentevidenceindicatesthatelevationofFAKactivityinhumancarcinomacellsisassociatedwithincreasedinvasivepotential.AcentralroleintumorformationandprogressionsuggestthatFAKisanattractivetargetfortherapeuticintervention.
      Cellsurfaceintegrinswhenengagedwiththeirligandsleadtotherecruitmentofanumberofintracellularproteinstospecializedsitesofthecytoplasmicfaceintofocaladhesions.Thesefocaladhesionplaquesarecomposedofmanyproteinsthatincludekinases,phosphatases,scaffoldingproteins,andG-proteins.Theactivationofintegrinsleadstothetyrosinephosphorylationofseveralcytoplasmicproteinscriticaltothebiochemicalprocess,includingFAK,Src,paxillin,tensin,andp130Cas.Thisclusteringandactivationofproteinsatthemembraneleadstotheactivationofmanydivergingsignalingpathwaysthatresultincytoskeletalre-organization.ThisstartswitheithertherecruitmentofSrcandFAKtotheplasmamembranefollowingintegrinorPI3Kinaseactivation.Thenon-receptorprotein-tyrosinekinases(PTKs)FAKandSrcarekeymembersofthefocaladhesioncomplex.ThesePTKsco-Localizeandassociatewiththetransmembraneintegrinsandvariousdownstreamtargetsofcertaingrowthfactorreceptors.TheoutcomeofSrcbindingtoFAKisthephosphorylationofseveraltyrosineresiduesinFAKanditsrelatedproteins,includingtheFAKautophosphorylationsiteTyr397.ThisautophosphorylationoccursimmediatelyafterintegrinclusteringandallowsfortheassociationofFAKwithvarioussignalingproteinssuchasPI3kinase,Grb2-Sos,p130Cas,andpaxillin.ThephosphorylationofFAKTyr397iscrucialformanyoftheestablishedBIOLOGicalrolesofFAK,includingcellmigration,cellcycleprogression,andpreventionofanoikis,aformofdetachment-inducedapoptosisandiscorrelatedwiththeinvasivenessoftumorsandisthoughttobeapivotalplayerbymodulatingtheturnoveroffocaladhesionstoregulatecellmigration.Phosphoinositide3-Kinase(PI3kinaseorPI3K)alsoplaysaroleincellmigrationviaitsbindingtotheautophosphorylationsite(Tyr397)ofFAKthroughoneorbothofitsSH2domainsinthep85subunitofPI3K.ThisisthesamesiteinFAKthatisboundbySrc.Intheory,thebindingofPI3kinasetoFAKmaybeinvolvedinactivitieslikecellproliferation,apoptosis,andmigrationthatarerelatedtothephosphorylationofTyr397.ThisissubstantiatedbytheobservationthatAKTisdownstreamofPI3kinaseandisamediatorinpreventingapoptosis.ThereforeitispossIBLethatFAK/PI3kinaseassociationmayhelpregulateapoptosis.
      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-FAKantibody:(PartNo.17-479A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • Anti-FAKdetectionantibody:(PartNo.17-479B)Onebottle(11mL)ofanti-FAKdetectionantibodycontainingsodiumazide,readytouse.
      • ELISADiluent:(PartNo.17-479C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 25XELISAWashBuffer:(PartNo.17-479D)Onebottle(50mL)of25XELISAWashBuffer.
      • Anti-RabbitIgGHRPconjugate:(PartNo.17-479E)Onevial(125µL)of100Xanti-rabbitHRPconjugatecontainingthimerosal.
      • HRPDiluent:(PartNo.17-479F)Onebottle(25mL)ofHRPDiluentcontainingthimerosal.
      • TMBSolution:(PartNo.17-479G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • StopSolution:(PartNo.17-479H)Onebottle(25mL)ofstopsolution,readytouse.
      • FAKStandard:(PartNo.17-479I)TwovialsofFAKstandard,lyophilized.
      • PlateCovers:Twoplatecovers.
      DetectionmethodChromogenic
      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
      ApplicationThisSTARFAKELISAKitisusedtomeasure&quantifyFAKlevels.
      KeyApplications
      • ELISA
      BiologicalInformation
      SpeciesReactivity
      • Human
      • Mouse
      • Rat
      AnalytesAvailable
      • FAK
      EntrezGeneNumber
      EntrezGeneSummaryThisgeneencodesacytoplasmicproteintyrosinekinasewhichisfoundconcentratedinthefocaladhesionsthatformbetweencellsgrowinginthepresenceofextracellularmatrixconstituents.TheencodedproteinisamemberoftheFAKsubfamilyofproteintyrosinekinasesbutlackssignificantsequencesimilaritytokinasesfromothersubfamilies.Activationofthisgenemaybeanimportantearlystepincellgrowthandintracellularsignaltransductionpathwaystriggeredinresponsetocertainneuralpeptidesortocellinteractionswiththeextracellularmatrix.Atleastfourtranscriptvariantsencodingfourdifferentisoformshavebeenfoundforthisgene,butthefull-lengthnaturesofonlytwoofthemhavebeendetermined.
      GeneSymbol
      • PTK2
      • pp125FAK
      • FADK
      • FAK1
      • FAK
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:Q05397#Non-receptorprotein-tyrosinekinaseimplicatedinsignalingpathwaysinvolvedincellmotility,proliferationandapoptosis.Activatedbytyrosine-phosphorylationinresponsetoeitherintegrinclusteringinducedbycelladhesionorantibodycross-linking,orviaG-proteincoupledreceptor(GPCR)occupancybyligandssuchasbombesinorlysophosphatidicacid,orviaLDLreceptoroccupancy.Playsapotentialroleinoncogenictransformationsresultinginincreasedkinaseactivity.
      SIZE:1052aminoacids;119233Da
      SUBUNIT:InteractswithCASfamilymembersandwithGIT1,SORBS1andBCAR3.InteractswithSHB(Bysimilarity).InteractswithTGFB1I1.
      SUBCELLULARLOCATION:Celljunction,focaladhesion;Cytoplasmicside.Note=Constituentoffocaladhesions.
      TISSUESPECIFICITY:Expressedinallorganstested,inlymphoidcelllines,butmostabundantlyinbrain.
      DOMAIN:SwissProt:Q05397ThefirstPro-richdomaininteractswiththeSH3domainofCRK-associatedsubstrate(BCAR1)andCASL(Bysimilarity).&Thecarboxy-terminalregionisthesiteoffocaladhesiontargeting(FAT)sequencewhichmediatesthelocalizationofFAK1tofocaladhesions.
      PTM:Phosphorylatedon6tyrosineresiduesuponactivation.
      SIMILARITY:Belongstotheproteinkinasesuperfamily.Tyrproteinkinasefamily.FAKsubfamily.&Contains1FERMdomain.&Contains1proteinkinasedomain.
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:1.6ng/mL
        RangeofDetection:1.6to100ng/mL
        SpeciesReactivity:Human,mouseandrat
      Dimensions
      MaterialsInformation
      MaterialsInformation
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