蔗糖密度梯度离心手册-sucrose gradient separation manual

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1、SucroseGradientSeparationManual_______________________AprotocoloptimizedfortheseparationofmitochondrialOXPHOSproteinsbydiscontinuoussucrosegradientseparation.March2005Alsoavailableatwww.mitosciences.com/PDF/sg.pdfwww.mitosciences.comsupport@mitosciences.comVer.#JM5.1ContentsPageI.Required

2、reagentsandequipment……………………………………2II.Samplepreparation……………………………………………………………3III.Samplesolubilization…………………………………………………………4IV.Sucrosegradientpreparation……………………………………………5V.Centrifugationofgradientsandelutionoffractions…………7VI.Sampleanalysis…………………………………………………………………8VII.Bufferrecipes………

3、…………………………………………………………….9VIII.Optimizationstepsandgeneraltips………………………………….10IX.TroubleshootingGuide………………………………………………………12XI.Flowchart……………………………………………………………………………13X.Notes………………………………………………………………………………….14I.RequiredReagentsŸn-dodecyl-b-D-maltopyranoside(Laurylmaltoside,Anatrace)ŸPhospha

4、tebufferedsaline,PBS(sectionVII)ŸSucrosesolutions15,20,25,27.5,30and35%ŸDoubledistilledwaterŸProteaseinhibitorcocktail(sectionVII)Ÿ13x51mmpolyallomercentrifugetubes(Beckman326819)RequiredEquipmentŸSwing-outcompatibleultracentrifugeandrotor(eg.BeckmanSW50.1ŸpHmeter,weighingbalanceandothers

5、tandardlabequipmentŸLaboratorybenchtopmicrofugeŸProteinelectrophoresisandWesternblottingequipment.2II.SamplepreparationTheMitoSciencessucrosegradientseparationprocedureisaproteinsubfractionationmethodoptimizedformitochondria.Itcanbeusedtoreducesamplecomplexityfacilitatinglargescaleproteom

6、icsefforts1(Tayloretal,Hansonetal).AlsowhenthisprocedurewascoupledtoMitoScienceshighlysensitiveWesternblottingantibodiesitallowsthedetectionofmis-assembledmitochondrialenzymecomplexesin2patientcelllines(Hansonetal).Thismethodresolvesasampleintoatleast10fractions.Itispossibletoseparatesolu

7、bilizedwholecellsintofractionsofmuchlowercomplexitybutwhenanalyzingalreadyisolatedmitochondriathefractionsareevenmoresimplified.Detailsofmitochondrialisolationcanbefoundatwww.mitosciences.com/PDF/mitos.pdfThetotalamountofOXPHOScomplexesinmitochondrialsamples/cellsam