Description | |
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CatalogueNumber | 17-479 |
BrandFamily | Upstate |
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Description | STARFAKELISAKit |
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BackgroundInformation | I.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. |
MaterialsRequiredbutNotDelivered | 1.Multi-channelorrepeatingPipettes 2.Plateshaker(optional) 3.Pipettorsandtipscapableofaccuratelymeasuring1-1000µL 4.GraduatedSEROlogicalpipettes 5.96-wellmicrotiterPlateReaderwith450nmfilter 6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata 7.Microfugetubesforstandardandsampledilutions 8.Mechanicalvortex 9.1litercontainer 10.Distilledordeionizedwater |
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Detectionmethod | Chromogenic |
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StorageConditions | Maintaintheunopenedkitat2-8°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. |
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Application | ThisSTARFAKELISAKitisusedtomeasure&quantifyFAKlevels. |
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Millipore常用产品报价单
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Millipore/475855-1R神奇滤布 密理博Millipore现货促销 | 475855-1R | Life Science Research | 膜 | 93.50 | Millipore/17-701/EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit | 17-701 | Life Science Research | 定量试剂盒 | 653.00 | Millipore/RIPAb+ Ago2 - RIP Validated Antibody and Primer Set | 03-110 | Life Science Research | 抗体套装 | 433.00 | Millipore/CBL468F | Anti-PECAM-1 Antibody, clone HC1/6, FITC conjugated/CBL468F/100 assays | CBL468F | Antibodies and Assays | 功能性抗体 | 298.00 | Millipore/ECM460 | αVβ3/PECAM Investigator Kit/ECM460/1 kit | ECM460 | Antibodies and Assays | 夹心法ELISA | 477.00 |