Description
CatalogueNumber	APT500
BrandFamily	Chemicon®
TradeName	CaspaTag Chemicon
Description	CaspaTagPan-CaspaseInSituAssayKit,Sulforhodamine
Overview	Apoptosisisanevolutionarilyconservedformofcellsuicide,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 ·DMSO

ProductInformation
Components	FLICAReagent(SR-VAD-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
Application	TheInSituFLICAPan-CaspaseDetectionKitforflowcytometryisafluorescent-basedassayfordetectionofactivecaspasesincellsundergoingapoptosis.
KeyApplications	FlowCytometry ActivityAssay

BIOLOGicalInformation
SpeciesReactivity	Mammals

PhysicochemicalInformation

Dimensions

MaterialsInformation

MaterialsInformation

Millipore 提供三种不同的 Immobilon PVDF 膜，是针对不同的蛋白质印迹应用的理想选择。 现在我们还 提供采用按规格裁切的印迹方片膜和两层过滤纸组成的印迹三明治。   与硝酸纤维素膜相比，Immobilon 膜具有多种优点。 它们不易破裂或卷 曲，便于裁剪且在切割过程中不会碎裂。 它们还有低背景、广泛溶剂相容性以及优异的着色能力。 此外，它们可以重复检测。 Immobilon-P 膜（沃比森-供应） ·         0.45μm 孔径 ·         建议用于大多数免疫印迹，特别在蛋 白质大于 20kDa 时 ·         Millipore 的快速免疫检测实验方法不需要膜封闭并缩短洗涤时间，将检测时间至少减少了两个小时，而不致降低灵敏 度或特异性 用于高通量实验室的印迹三明治 ·         当您需要处理多个印迹时，印迹三明治是一种便利省时的选择。 我们的印迹三明 治包含多片 Immobilon-P 膜，其中插入了按规格裁切的多张层析级印迹纸。 转印滤纸 ·         按照大多数免疫印迹尺寸进行裁切的层析级转印滤纸。 Immobilon-PSQ 膜（沃比森-供应） ·         0.2μm 孔径和大的内表面面积 ·         较其它膜具有更高的 蛋白质吸附率和测序得率 ·         建议用于小于 20kDa 的印迹蛋白质 ·         防止小分子量的蛋白质的渗漏 Immobilon-FL 膜（沃比森-供应） ·        针对荧光应用进行优化的转印膜 ·         极低的背景提高了所 有荧光检测实验的灵敏度 ·         与所有常用的荧光探针（所有激发和发射波长）兼容 ·         是多态性检测和化学荧光应用的理想选择