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Millipore/APT400 | CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein/APT400/100 assays
  • Millipore/APT400 | CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein/APT400/100 assays

Millipore/APT400 | CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein/APT400/100 assays

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貨號(hào): APT400
品牌: Millipore
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    • Description
      CatalogueNumberAPT400
      BrandFamilyChemicon®
      TradeName
      • CaspaTag
      • Chemicon
      DescriptionCaspaTagPan-CaspaseInSituAssayKit,Fluorescein
      OverviewApoptosisisanevolutionarilyconservedformofcellsuicide,whichfollowsaspecializedcellularprocess.Thecentralcomponentofthisprocessisacascadeofproteolyticenzymescalledcaspases.Theseenzymesparticipateinaseriesofreactionsthataretriggeredinresponsetopro-apoptoticsignalsandresultinthecleavageofproteinsubstrates,causingthedisassemblyofthecell(Sleeetal.1999).

      CaspaseshavebeenidentifiedinorganismsrangingfromC.eleganstohumans.Themammaliancaspasesplaydistinctrolesinapoptosisandinflammation.Inapoptosis,caspasesareresponsIBLeforproteolyticcleavagesthatleadtocelldisassembly(effectorcaspases),andareinvolvedinupstreamregulatoryevents(initiatorcaspases).Anactivecaspaseconsistsoftwolargeandtwosmallsubunitsthatformtwoheterodimerswhichassociateinatetramer(Walkeretal.1994;Wilsonetal.1994;Rotondaetal.1996).Incommonwithotherproteases,caspasesaresynthesizedasprecursorsthatundergoproteolyticmaturation,eitherautocatalyticallyorinacascadebyenzymeswithsimilarspecificity(Kumar1999).

      Caspaseenzymesspecificallyrecognizea4or5aminoacidsequenceonthetargetsubstratewhichnecessarilyincludesanasparticacidresidue.Thisresidueisthetargetforcleavage,whichoccursatthecarbonylendoftheasparticacidresidue(Thornberryetal.1997).Caspasescanbedetectedviaimmunoprecipitation,immunoblottingtechniquesusingcaspasespecificantibodies,orbyemployingFluorochromesubstrateswhichbecomefluorescentuponcleavagebythecaspase.

      TestPrinciple:

      CHEMICON"sInSituCaspaseDetectionKitsuseanovelapproachtodetectactivecaspases.ThemethodologyisbasedonFluorochromeInhibitorsofCaspases(FLICA).Theinhibitorsarecellpermeableandnon-cytotoxic.Onceinsidethecell,theinhibitorbindscovalentlytotheactivecaspase(Ekertetal.1999).Thiskitusesacarboxyfluorescein-labeledfluoromethylketonepeptideinhibitorofcaspase(FAM-VAD-FMK),whichproducesagreenfluorescence.Whenaddedtoapopulationofcells,theFAM-VAD-FMKprobeenterseachcellandcovalentlybindstoareactivecysteineresiduethatresidesonthelargesubunitoftheactivecaspaseheterodimer,therebyinhibitingfurtherenzymaticactivity.Theboundlabeledreagentisretainedwithinthecell,whileanyunboundreagentwilldiffuseoutofthecellandiswashedaway.Thegreenfluorescentsignalisadirectmeasureoftheamountofactivecaspasepresentinthecellatthetimethereagentwasadded.Cellsthatcontaintheboundlabeledreagentcanbeanalyzedby96-wellplate-basedfluorometry,fluorescencemicroscopy,orflowcytometry.

      Application:

      TheCHEMICONInSituFLICAPan-CaspaseDetectionKitisafluorescent-basedassayfordetectionofactivecaspasesincellsundergoingapoptosis.Thekitisforresearchuseonly.Notforuseindiagnosticortherapeuticprocedures.
      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-VAD-FMK):Fourlyophilizedvials
      • 10XWashBuffer:60mL
      • Fixative(10%formaldehyde):6mL
      • PropidiumIodide:1mLat250μg/mL,ready-to-use
      • HoechstStain:1mLat200μg/mL,ready-to-use
      DetectionmethodFluorescent
      StorageandShippingInformation
      StorageConditions·Storeunopenedkitmaterialsat2-8°Cuptotheirexpirationdate.

      ·ReconstitutedFLICAReagent(150X)shouldbefrozenat-20oCforupto6months,andmaybethawedtwiceduringthistime.Aliquotintoseparateambertubesifdesired.Protectfromlightatalltimes.

      ·Storediluted(1X)washbufferupto-20oCfor2weeks.
      Applications
      ApplicationTheInSituFLICAPan-CaspaseDetectionKitforflowcytometryisafluorescent-basedassayfordetectionofactivecaspasesincellsundergoingapoptosis.
      KeyApplications
      • FlowCytometry
      • ActivityAssay
      BIOLOGicalInformation
      SpeciesReactivity
      • Mammals
      EntrezGeneNumber
      EntrezGeneSummaryThisgeneencodesaproteinwhichisamemberofthecysteine-asparticacidprotease(caspase)family.Sequentialactivationofcaspasesplaysacentralroleintheexecution-phaseofcellapoptosis.Caspasesexistasinactiveproenzymeswhichundergoproteolyticprocessingatconservedasparticresiduestoproducetwosubunits,largeandsmall,thatdimerizetoformtheactiveenzyme.Thisproteincleavesandactivatescaspases3and7,andtheproteinitselfisprocessedbycaspase8.MutationsinthisgeneareassociatedwithapoptosisdefectsseenintypeIIautoimmunelymphoproliferativesyndrome.Threealternativelysplicedtranscriptvariantsencodingdifferentisoformshavebeendescribedforthisgene.
      GeneSymbol
      • CASP1
      • IL1BCE
      • P45
      • IL-1BC
      • IL1BC
      • p45
      • IL1B-convertase
      • CASP-1
      • ICE
      • EC3.4.22.36[Contains:Caspase-1subunitp20
      • Caspase-1subunitp10].
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:Q92851#IsoformCisproteolyticallyinactive.
      SIZE:521aminoacids;58951Da
      SUBUNIT:Heterotetramerthatconsistsoftwoanti-parallelarrangedheterodimers,eachoneformedbya23/17kDa(p23/17)(dependingonthesplicingevents)anda12kDa(p12)subunit(Bysimilarity).Self-associates.InteractswithFADDandCASP8.FoundinaFassignalingcomplexconsistingofFAS,FADD,CASP8andCASP10.
      TISSUESPECIFICITY:Detectableinmosttissues.Lowestexpressionisseeninbrain,kidney,prostate,testisandcolon.
      PTM:CleavagebygranzymeBandautocatalyticactivitygeneratethetwoactivesubunits.&PhosphorylateduponDNAdamage,probablybyATMorATR.
      DISEASE:SwissProt:Q92851#DefectsinCASP10arethecauseofautoimmunelymphoproliferativesyndrometype2A(ALPS2A)[MIM:603909].ALPS2ischaracterizedbyabnormallymphocyteanddendriticcellhomeostasisandimmuneregulatorydefects.&DefectsinCASP10areacauseoffamilialnon-Hodgkinlymphoma(NHL)[MIM:605027].NHLisacancerthatstartsincellsofthelymphsystem,whichispartofthebody"simmunesystem.NHLscanoccuratanyageandareoftenmarkedbyenlargedlymphnodes,feverandweightloss.&DefectsinCASP10areacauseofgastriccancers[MIM:137215].
      SIMILARITY:SwissProt:Q92851##BelongstothepeptidaseC14family.&Contains2DED(deatheffector)domains.
      PhysicochemicalInformation
      Dimensions
      MaterialsInformation
      MaterialsInformation
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