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Millipore/APT408 | CaspaTag Caspase 8 In Situ Assay Kit, Fluorescein/APT408/100 assays
  • Millipore/APT408 | CaspaTag Caspase 8 In Situ Assay Kit, Fluorescein/APT408/100 assays

Millipore/APT408 | CaspaTag Caspase 8 In Situ Assay Kit, Fluorescein/APT408/100 assays

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貨號(hào): APT408
品牌: Millipore
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    • Description
      CatalogueNumberAPT408
      BrandFamilyChemicon®
      TradeName
      • CaspaTag
      • Chemicon
      DescriptionCaspaTagCaspase8InSituAssayKit,Fluorescein
      BackgroundInformationApoptosisisanevolutionarilyconservedformofcellsuicide,whichfollowsaspecializedcellularprocess.Thecentralcomponentofthisprocessisacascadeofproteolyticenzymescalledcaspases.Theseenzymesparticipateinaseriesofreactionsthataretriggeredinresponsetopro-apoptoticsignalsandresultinthecleavageofproteinsubstrates,causingthedisassemblyofthecell1.

      CaspaseshavebeenidentifiedinorganismsrangingfromC.eleganstohumans.Themammaliancaspasesplaydistinctrolesinapoptosisandinflammation.Inapoptosis,caspasesareresponsIBLeforproteolyticcleavagesthatleadtocelldisassembly(effectorcaspases),andareinvolvedinupstreamregulatoryevents(initiatorcaspases).Anactivecaspaseconsistsoftwolargeandtwosmallsubunitsthatformtwoheterodimers,whichassociateinatetramer2-4.Incommonwithotherproteases,caspasesaresynthesizedasprecursorsthatundergoproteolyticmaturation,eitherautocatalyticallyorinacascadebyenzymeswithsimilarspecificity5.

      Caspaseenzymesspecificallyrecognizea4or5aminoacidsequenceonthetargetsubstrate,whichnecessarilyincludesanasparticacidresidue.Thisresidueisthetargetforcleavage,whichoccursatthecarbonylendoftheasparticacidresidue6.Caspasescanbedetectedviaimmunoprecipitation,immunoblottingtechniquesusingcaspasespecificantibodies,orbyemployingFluorochromesubstrates,whichbecomefluorescentuponcleavagebythecaspase.
      MaterialsRequiredbutNotDelivered·Culturedcellswithmedia

      ·Reagentstoinduceapoptosis

      ·15mLpolystyrenecentrifugetubes

      ·Ambervialsortubesforstorageof150Xconcentrateat-20°C

      ·600mLgraduatedcylinder

      ·Microscopeslides

      ·Hemocytometer

      ·Centrifuge(400xg)

      ·37°Cincubator

      ·Vortexer

      ·Adjustablevolumepipettorwithdisposabletips

      ·Deionizedwater

      ·PBS,pH7.4

      ·DMSO
      ProductInformation
      Components
      • FLICAReagent(FAM-LETD-FMK):Fourlyophilizedvials
      • 10XWashBuffer:60mL
      • Fixative:6mL
      • PropidiumIodide:1mLat250μg/mL,ready-to-use
      • HoechstStain:1mLat200μg/mL,ready-to-use
      DetectionmethodFluorescent
      StorageandShippingInformation
      StorageConditions·Storeunopenedkitmaterialsat2-8°Cuptotheirexpirationdate.

      ·ReconstitutedFLICAReagent(150X)shouldbefrozenat-20°Cforupto6months,andmaybethawedtwiceduringthistime.Aliquotintoseparateambertubesifdesired.Protectfromlightatalltimes.

      ·Storediluted(1X)washbufferupto-20°Cfor2weeks.
      Applications
      ApplicationTheCaspaTagCaspase-8InSituAssayKit,Fluoresceinforflowcytometryisafluorescent-basedassayfordetectionofactivecaspase-8incellsundergoingapoptosis.
      KeyApplications
      • FlowCytometry
      • ActivityAssay
      ApplicationNotesTheCHEMICON?CaspaTag?Caspase-8InSituAssayKit,Fluoresceinisafluorescent-basedassayfordetectionofactivecaspase-8incellsundergoingapoptosis.Thekitisforresearchuseonly.Notforuseindiagnosticortherapeuticprocedures.

      TestPrinciple

      CHEMICON?"sCaspaTag?Caspase-8InSituAssayKitsuseanovelapproachtodetectactivecaspases.ThemethodologyisbasedonFluorochromeInhibitorsofCaspases(FLICA).Theinhibitorsarecellpermeableandnon-cytotoxic.Onceinsidethecell,theinhibitorbindscovalentlytotheactivecaspase7.Thiskitusesacarboxyfluorescein-labeledfluoromethylketonepeptideinhibitorofcaspase-8(FAM-LETD-FMK),whichproducesagreenfluorescence.Whenaddedtoapopulationofcells,theFAM-LETD-FMKprobeenterseachcellandcovalentlybindstoareactivecysteineresiduethatresidesonthelargesubunitoftheactivecaspaseheterodimer,therebyinhibitingfurtherenzymaticactivity.Theboundlabeledreagentisretainedwithinthecell,whileanyunboundreagentwilldiffuseoutofthecellandiswashedaway.Thegreenfluorescentsignalisadirectmeasureoftheamountofactivecaspase-8presentinthecellatthetimethereagentwasadded.Cellsthatcontaintheboundlabeledreagentcanbeanalyzedby96-wellplate-basedfluorometry,fluorescencemicroscopy,orflowcytometry.
      BIOLOGicalInformation
      EntrezGeneNumber
      EntrezGeneSummaryThisgeneencodesamemberofthecysteine-asparticacidprotease(caspase)family.Sequentialactivationofcaspasesplaysacentralroleintheexecution-phaseofcellapoptosis.Caspasesexistasinactiveproenzymescomposedofaprodomain,alargeproteasesubunit,andasmallproteasesubunit.Activationofcaspasesrequiresproteolyticprocessingatconservedinternalasparticresiduestogenerateaheterodimericenzymeconsistingofthelargeandsmallsubunits.ThisproteinisinvolvedintheprogrammedcelldeathinducedbyFasandvariousapoptoticstimuli.TheN-terminalFADD-likedeatheffectordomainofthisproteinsuggeststhatitmayinteractwithFas-interactingproteinFADD.ThisproteinwasdetectedintheinsolublefractionoftheaffectedbrainregionfromHuntingtondiseasepatientsbutnotinthosefromnormalcontrols,whichimplicatedtheroleinneurodegenerativediseases.Manyalternativelysplicedtranscriptvariantsencodingdifferentisoformshavebeendescribed,althoughnotallvariantshavehadtheirfull-lengthsequencesdetermined.
      GeneSymbol
      • CASP8
      • MCH5
      • MGC78473
      • CASP-8
      • MACH
      • ALPS2B
      • procaspase-8
      • FLICE
      • CAP4
      • EC3.4.22.61[Contains:Caspase-8subunitp18
      • Caspase-8subunitp10].
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:Q14790#MostupstreamproteaseoftheactivationcascadeofcaspasesresponsiblefortheTNFRSF6/FASmediatedandTNFRSF1Ainducedcelldeath.BindingtotheadaptermoleculeFADDrecruitsittoeitherreceptor.Theresultingaggregatecalleddeath-inducingsignalingcomplex(DISC)performsCASP8proteolyticactivation.TheactivedimericenzymeisthenliberatedfromtheDISCandfreetoactivatedownstreamapoptoticproteases.ProteolyticfragmentsoftheN-terminalpropeptide(termedCAP3,CAP5andCAP6)arelikelyretainedintheDISC.CleavesandactivatesCASP3,CASP4,CASP6,CASP7,CASP9andCASP10.MayparticipateintheGZMBapoptoticpathways.CleavesADPRT.Hydrolyzesthesmall-moleculesubstrate,Ac-Asp-Glu-Val-Asp--AMC.LikelytargetforthecowpoxvirusCRMAdeathinhibitoryprotein.Isoforms5,6,7and8lackthecatalyticsiteandmayinterferewiththepro-apoptoticactivityofthecomplex.
      SIZE:479aminoacids;55391Da
      SUBUNIT:Heterotetramerthatconsistsoftwoanti-parallelarrangedheterodimers,eachoneformedbya18kDa(p18)anda10kDa(p10)subunit.InteractswithFADD,CFLARandPEA15.Isoform9interactsattheendoplasmicreticulumwithacomplexcontainingBCAP31,BAP29,BCL2and/orBCL2L1.
      SUBCELLULARLOCATION:Cytoplasm.
      TISSUESPECIFICITY:Isoforms1,5and7areexpressedinawidevarietyoftissues.Highestexpressioninperipheralbloodleukocytes,spleen,thymus,andliver.Barelydetectableinbrain,testis,andskeletalmuscle.DOMAIN:SwissProt:Q14790Isoform9containsaN-terminalextensionthatisrequiredforinteractionwiththeBCAP31complex.
      PTM:Generationofthesubunitsrequiresassociationwiththedeath-inducingsignalingcomplex(DISC),whereasadditionalprocessingislikelyduetotheautocatalyticactivityoftheactivatedprotease.GZMBandCASP10canbeinvolvedintheseprocessingevents.&PhosphorylateduponDNAdamage,probablybyATMorATR.
      DISEASE:SwissProt:Q14790#DefectsinCASP8arethecauseofcaspase-8deficiency(CASP8D)[MIM:607271].CASP8Disadisorderresemblingautoimmunelymphoproliferativesyndrome(ALPS).Itischaracterizedbylymphadenopathy,splenomegaly,anddefectiveCD95-inducedapoptosisofperipheralbloodlymphocytes(PBLs).ItleadstodefectsinactivationofT-lymphocytes,B-lymphocytes,andnaturalkillercellsleADIngtoimmunodeficiencycharacterizedbyrecurrentsinopulmonaryandherpessimplexvirusinfectionsandpoorresponsestoimmunization.
      SIMILARITY:BelongstothepeptidaseC14family.&Contains2DED(deatheffector)domains.
      PhysicochemicalInformation
      Dimensions
      MaterialsInformation
      MaterialsInformation
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