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Millipore/APT523 | CaspaTag Caspase 3,7 In Situ Assay Kit, Sulforhodamine/APT523/25 assays
  • Millipore/APT523 | CaspaTag Caspase 3,7 In Situ Assay Kit, Sulforhodamine/APT523/25 assays

Millipore/APT523 | CaspaTag Caspase 3,7 In Situ Assay Kit, Sulforhodamine/APT523/25 assays

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貨號(hào): APT523
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    • Description
      CatalogueNumberAPT523
      BrandFamilyChemicon®
      TradeName
      • CaspaTag
      • Chemicon
      DescriptionCaspaTagCaspase3,7InSituAssayKit,Sulforhodamine
      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.
      ProductInformation
      DetectionmethodFluorescent
      StorageandShippingInformation
      StorageConditions·Storeunopenedkitmaterialsat2-8°Cuptotheirexpirationdate.

      ·ReconstitutedCaspaTag?reagent(150X)shouldbefrozenat-20°Cforupto6months,andmaybethawedtwiceduringthistime.Aliquotintoseparateambertubesifdesired.Protectfromlightatalltimes.

      ·Storediluted(1X)washbufferupto-20°Cfor2weeks.
      Applications
      ApplicationTheCaspaTagCaspase3,7InSituAssayKitforflowcytometryisafluorescent-basedassayfordetectionofactivecaspases-3&-7incellsundergoingapoptosis.
      KeyApplications
      • FlowCytometry
      • ActivityAssay
      • Immunofluorescence
      ApplicationNotesTheCHEMICON?CaspaTag?Caspase3,7InSituAssayKitisafluorescent-basedassayfordetectionofactivecaspases-3and-7incellsundergoingapoptosis.Thekitisforresearchuseonly.Notforuseindiagnosticortherapeuticprocedures.

      TestPrinciple

      CHEMICON?"sCaspaTag?Caspase-3,7InSituAssayKitsuseanovelapproachtodetectactivecaspases.ThemethodologyisbasedonFluorochromeInhibitorsofCaspases(FLICA).Theinhibitorsarecellpermeableandnon-cytotoxic.Onceinsidethecell,theinhibitorbindscovalentlytotheactivecaspase7.Thiskitusesacarboxyfluorescein-labeledfluoromethylketonepeptideinhibitorofcaspases-3and-7(SR-DEVD-FMK),whichproducesaredfluorescence.Whenaddedtoapopulationofcells,theSR-DEVD-FMKprobeenterseachcellandcovalentlybindstoareactivecysteineresiduethatresidesonthelargesubunitoftheactivecaspaseheterodimer,therebyinhibitingfurtherenzymaticactivity.Theboundlabeledreagentisretainedwithinthecell,whileanyunboundreagentwilldiffuseoutofthecellandiswashedaway.Theredfluorescentsignalisadirectmeasureoftheamountofactivecaspases-3and-7presentinthecellatthetimethereagentwasadded.Cellsthatcontaintheboundlabeledreagentcanbeanalyzedby96-wellplate-basedfluorometry,fluorescencemicroscopy,orflowcytometry.
      BIOLOGicalInformation
      SpeciesReactivity
      • Mammals
      EntrezGeneNumber
      EntrezGeneSummaryThisgeneencodesamemberofthecysteine-asparticacidprotease(caspase)family.Sequentialactivationofcaspasesplaysacentralroleintheexecution-phaseofcellapoptosis.Caspasesexistasinactiveproenzymescomposedofaprodomain,alargeproteasesubunit,andasmallproteasesubunit.Activationofcaspasesrequiresproteolyticprocessingatconservedinternalasparticresiduestogenerateaheterodimericenzymeconsistingofthelargeandsmallsubunits.ThisproteinisinvolvedintheprogrammedcelldeathinducedbyFasandvariousapoptoticstimuli.TheN-terminalFADD-likedeatheffectordomainofthisproteinsuggeststhatitmayinteractwithFas-interactingproteinFADD.ThisproteinwasdetectedintheinsolublefractionoftheaffectedbrainregionfromHuntingtondiseasepatientsbutnotinthosefromnormalcontrols,whichimplicatedtheroleinneurodegenerativediseases.Manyalternativelysplicedtranscriptvariantsencodingdifferentisoformshavebeendescribed,althoughnotallvariantshavehadtheirfull-lengthsequencesdetermined.
      GeneSymbol
      • CASP3
      • apopain
      • CASP-3
      • procaspase3
      • CPP32B
      • SCA-1
      • CPP-32
      • Apopain
      • CPP32
      • Yama
      • EC3.4.22.56[Contains:Caspase-3subunitp17
      • Caspase-3subunitp12].
      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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