首页 > Millipore > Millipore/APT500 | CaspaTag Pan-Caspase In Situ Assay Kit, Sulforhodamine/APT500/100 assays

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Millipore/APT500 | CaspaTag Pan-Caspase In Situ Assay Kit, Sulforhodamine/APT500/100 assays

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DescriptionCatalogueNumberAPT500BrandFamilyChemicon®TradeNameCaspaTagChemiconDescriptionCaspaTagPan-CaspaseInSituAssayKit,SulforhodamineOverviewApoptosisisanevolutionarilyconservedformofcellsuicide,whichfollowsaspecializedcellularprocess.Thecentralcomponentofthisprocessisacascadeofproteolyticenzymescalledcaspases.Theseenzymesparticipateinaseriesofreactionsthataretriggeredinresponsetopro-apoptoticsignalsandresultinthecleavageofproteinsubstrates,causingthedisassemblyofthecell(Arends&Wyllie1991).CaspaseshavebeenidentifiedinorganismsrangingfromC.eleganstohumans.Themammaliancaspasesplaydistinctrolesinapoptosisandinflammation.Inapoptosis,caspasesareresponsIBLeforproteolyticcleavagesthatleadtocelldisassembly(effectorcaspases),andareinvolvedinupstreamregulatoryevents(initiatorcaspases).Anactivecaspaseconsistsoftwolargeandtwosmallsubunitsthatformtwoheterodimerswhichassociateinatetramer(Sleeetal.1999;Salvesen&Dixit1997;Alnemrietal.1996).Incommonwithotherproteases,caspasesaresynthesizedasprecursorsthatundergoproteolyticmaturation,eitherautocatalyticallyorinacascadebyenzymeswithsimilarspecificity(Sleathetal.1990).Caspaseenzymesspecificallyrecognizea4or5aminoacidsequenceonthetargetsubstratewhichnecessarilyincludesanasparticacidresidue.Thisresidueisthetargetforcleavage,whichoccursatthecarbonylendoftheasparticacidresidue(Thornberryetal.1992).Caspasescanbedetectedviaimmunoprecipitation,immunoblottingtechniquesusingcaspasespecificantibodies,orbyemployingFluorochromesubstrateswhichbecomefluorescentuponcleavagebythecaspase.TestPrinciple:CHEMICON"sInSituCaspaseDetectionKitsuseanovelapproachtodetectactivecaspases.ThemethodologyisbasedonFluorochromeInhibitorsofCaspases(FLICA).Theinhibitorsarecellpermeableandnon-cytotoxic.Onceinsidethecell,theinhibitorbindscovalentlytotheactivecaspase(Rotondaetal.1996).Thiskitusesasulforhodamine-labeledfluoromethylketonepeptideinhibitorofcaspase(SR-VAD-FMK),whichproducesaredfluorescence.Whenaddedtoapopulationofcells,theSR-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·DMSOProductInformationComponentsFLICAReagent(SR-VAD-FMK):Fourlyophilizedvials10XWashBuffer:60mLFixative:6mLHoechstStain:1mLat200μg/mL,ready-to-useDetectionmethodFluorescentStorageandShippingInformationStorageConditions·Storeunopenedkitmaterialsat2-8°Cuptotheirexpirationdate.·ReconstitutedFLICAReagent(150X)shouldbefrozenat-20ºCforupto6months,andmaybethawedtwiceduringthistime.Aliquotintoseparateambertubesifdesired.Protectfromlightatalltimes.·Storediluted(1X)washbufferupto-20ºCfor2weeks.ApplicationsApplicationTheInSituFLICAPan-CaspaseDetectionKitforflowcytometryisafluorescent-basedassayfordetectionofactivecaspasesincellsundergoingapoptosis.KeyApplicationsFlowCytometryActivityAssayBIOLOGicalInformationSpeciesReactivityMammalsPhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation

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