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    當前位置 ： Millipore >>> 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

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

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    貨號: 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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