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1、Nuclear-CytosolExtractionKit(100assays,5-10´106cellsperassay>#P1200ThisNuclear-CytosolExtractionKitprovidesacompletesystemforpreparingbothnuclearandcytoplasmicproteinextractsfrommammaliantissueorculturedcells.Thekitprovidesflexibilityforoptimalproteinextraction,forexample,thechoic
2、ebetweendetergentandnon-detergentextraction.Withtheoptimizedreagentsandprocedures,theextractscanbepreparedinlessthan1hourandarehighlypuresincethereislittleornocross-contaminationbetweennuclearandcytoplasmicextracts.Theextractednuclearandcytoplasmicproteinfractionscanbeusedindownst
3、reamassayssuchastranscriptionalactivity,gelshiftassay,reporterassays,enzymeactivityassays,immunoprecipitation,andWesternblotting.b5E2RGbCAPKitContentsandStorageCytosolExtractionBufferA(CEB-A>50ml,4oC。CytosolExtractionBufferB(CEB-B>2x1.5ml,4oCp1EanqFDPwNuclearExtractionBuffer(NEB>1
4、0ml,4oC.Thekitissufficientfor100extractionsofeach5´106cells.DXDiTa9E3dThekitcompositionsarestableatleastfor1yearat4oC.Notethatproteaseinhibitorsarenotincludedinbuffer.Usercanaddproteaseinhibitors,althoughusuallyallbuffersworkfinewithoutadditionoftheinhibitors.RTCrpUDGiTGeneralCons
5、iderationKeepallbuffersoniceandhandlethesampleoniceorat4oC.5PCzVD7HxAEstimationofpackedcellvolume(PCV>:PCVisanestimatedvolumeofthepelletedcellsaftercentrifugation.jLBHrnAILgThevolumeofPCVisdependentoncelltypes.The1x106packedcellsusuallygivea10-20mlPCV.Theprotocolisdescribedforfrac
6、tionationofupto5~10x106.Iflessormorecellsareextracted,proportionallyscaleuptheinitialvolumeoftheextractionbuffers.xHAQX74J0XCellNumbers5x1051x1065x106EstimatedPackedCellVolume(PCV,ml>510-2060-100LDAYtRyKfECytosolExtractionBufferA(ml>2550-100300-500Zzz6ZB2LtkCytosolExtractionBuffer
7、B(ml>1.2-1.53-618-30dvzfvkwMI1NuclearExtractionBuffer(ml>5-1020100Nuclear/CytosolFractionationProtocol:Collectcellsa.Adherentcells:Whencellsreach70-90%confluence,removetheculturemedium.RinsethecellswithPBS.rqyn14ZNXIOptional:Add5-10mlofNon-enzymeCellDisassociationSolution(#C1000>t
8、odetachthecells.Incubateatroomtem
