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Millipore/APT503 | CaspaTag Caspase 3,7 In Situ Assay Kit, Sulforhodamine/APT503/100 assays

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貨號: APT503

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Description
CatalogueNumber	APT503
BrandFamily	Chemicon®
TradeName	CaspaTag Chemicon
Description	CaspaTagCaspase3,7InSituAssayKit,Sulforhodamine
Overview	Apoptosisisanevolutionarilyconservedformofcellsuicide,whichfollowsaspecializedcellularprocess.Thecentralcomponentofthisprocessisacascadeofproteolyticenzymescalledcaspases.Theseenzymesparticipateinaseriesofreactionsthataretriggeredinresponsetopro-apoptoticsignalsandresultinthecleavageofproteinsubstrates,causingthedisassemblyofthecell1. CaspaseshavebeenidentifiedinorganismsrangingfromC.eleganstohumans.Themammaliancaspasesplaydistinctrolesinapoptosisandinflammation.Inapoptosis,caspasesareresponsIBLeforproteolyticcleavagesthatleadtocelldisassembly(effectorcaspases),andareinvolvedinupstreamregulatoryevents(initiatorcaspases).Anactivecaspaseconsistsoftwolargeandtwosmallsubunitsthatformtwoheterodimerswhichassociateinatetramer2-4.Incommonwithotherproteases,caspasesaresynthesizedasprecursorsthatundergoproteolyticmaturation,eitherautocatalyticallyorinacascadebyenzymeswithsimilarspecificity5. Caspaseenzymesspecificallyrecognizea4or5aminoacidsequenceonthetargetsubstratewhichnecessarilyincludesanasparticacidresidue.Thisresidueisthetargetforcleavage,whichoccursatthecarbonylendoftheasparticacidresidue6.Caspasescanbedetectedviaimmunoprecipitation,immunoblottingtechniquesusingcaspasespecificantibodies,orbyemployingFluorochromesubstrateswhichbecomefluorescentuponcleavagebythecaspase. TestPrinciple: CHEMICON"sInSituCaspaseDetectionKitsuseanovelapproachtodetectactivecaspases.ThemethodologyisbasedonFluorochromeInhibitorsofCaspases(FLICA).Theinhibitorsarecellpermeableandnon-cytotoxic.Onceinsidethecell,theinhibitorbindscovalentlytotheactivecaspase7.Thiskitusesacarboxyfluorescein-labeledfluoromethylketonepeptideinhibitorofcaspase-3(SR-DEVD-FMK),whichproducesagreenfluorescence.Whenaddedtoapopulationofcells,theSR-DEVD-FMKprobeenterseachcellandcovalentlybindstoareactivecysteineresiduethatresidesonthelargesubunitoftheactivecaspaseheterodimer,therebyinhibitingfurtherenzymaticactivity.Theboundlabeledreagentisretainedwithinthecell,whileanyunboundreagentwilldiffuseoutofthecellandiswashedaway.Thegreenfluorescentsignalisadirectmeasureoftheamountofactivecaspase-3presentinthecellatthetimethereagentwasadded.Cellsthatcontaintheboundlabeledreagentcanbeanalyzedby96-wellplate-basedfluorometry,fluorescencemicroscopy,orflowcytometry. Application: TheCHEMICONInSituFLICACaspase-3DetectionKitisafluorescent-basedassayfordetectionofactivecaspase-3incellsundergoingapoptosis.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(SR-DEVD-FMK):Fourlyophilizedvials 10XWashBuffer:60mL Fixative:6mL HoechstStain:1mLat200μg/mL,ready-to-use
Detectionmethod	Fluorescent

StorageandShippingInformation
StorageConditions	·Storeunopenedkitmaterialsat2-8°Cuptotheirexpirationdate. ·ReconstitutedFLICAReagent(150X)shouldbefrozenat-20oCforupto6months,andmaybethawedtwiceduringthistime.Aliquotintoseparateambertubesifdesired.Protectfromlightatalltimes. ·Storediluted(1X)washbufferupto-20oCfor2weeks.

Applications
KeyApplications	FlowCytometry ActivityAssay Immunofluorescence

BIOLOGicalInformation
SpeciesReactivity	Mammals
EntrezGeneNumber	NM_004346.3 NM_032991.2
EntrezGeneSummary	Thisgeneencodesamemberofthecysteine-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	P42574 Q14790
UniProtSummary	FUNCTION: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.

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Millipore/APT503 | CaspaTag Caspase 3,7 In Situ Assay Kit, Sulforhodamine/APT503/100 assays

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