INSTRUCTIONSPierceBiotechnologyPOBox117(815)968-0747www.thermoscientific.com/pierce3747N.MeridianRoadRockford,lL61105USA(815)968-7316faxNumberDescription89874MitochondriaIsolationKitforCulturedCells,containssufficientreagentstoisolateintactmitochondriafrom50pelletsofculturedmammaliancellscontaining2×107cellseachKitContents:MitochondriaIsolationReagentA,50mLMitochondriaIsolationReagentB,500µLMitochondriaIsolationReagentC,65mLStorage:Uponreceiptstorekitat4°C.Productisshippedatambienttemperature.IntroductionTheThermoScientificMitochondriaIsolationKitforCulturedCellsenablesisolationofintactmitochondriafromculturedmammaliancells.Thekitofferstwooptionsfortheseparationofmitochondriafromcytosoliccomponents(Figure1).Oneoptionisauniqueprotocolutilizingareagent-basedmethodallowingmultiplesamplestobeprocessedconcurrently.ThesecondoptionusestraditionalDouncehomogenizationandprovidesapproximatelytwo-foldmoremitochondria,basedonproteinanalysis.Bothprotocolsrelyondifferentialcentrifugationtoseparatethemitochondrialandcytosolicfractionswithabench-topmicrocentrifugeandarecompletedinapproximately40minutes(post-cellharvest).Eachprocedurehasbeenoptimizedformaximumyieldofmitochondriawithminimaldamagetointegrity.Onceisolated,themitochondriamaybeusedforanumberofdownstreamapplications,includingapoptosis,signaltransductionandmetabolicstudies.Add800µLReagentAtocells(20x106)Incubate2minutesoniceOptionAAdd10µLReagentB.Incubate5minutesonice,vortexingeveryminuteOptionBDouncehomogenizeAdd800µLReagentCCentrifugeat700×gfor10minutesat4°CCollectsupernatantandcentrifuge*at12,000×gfor15minutesat4°CRemovesupernatant.Washpellet(mitochondria)with500µLofReagentCCentrifugeat12,000×gfor5minutesat4°C*Toobtainamorepurifiedpreparationofmitochondria,thisstepmaybeperformedat3,000×g.Figure1.ProceduresummaryofthePierceMitochondriaIsolationKit.ImportantProductInformation•Thiskitallowstwooptionsformitochondriaisolation:areagent-basedmethodorDouncehomogenization.Thekitcontainssufficientreagentsforatot...
