• 20mlglasshomogenizerequippedwithTeflonpestle
• HomogenizerorDrillpress
• Superspeedcentrifuge(SorvallRC-5B)withtheequivalentofSorvallSS-34rotors
• 50mlpolycarbonatecentrifugetubes(Sorvall#03146)
• Ultracentrifuge(BeckmanL7-55)withtheequivalentsofBeckman50.2TiandSW-28rotors
• 31.5mlthick-walledpolycarbonateultracentrifugetubes(Beckman#336091)withscrewcaps(Beckman#338906)
• 25mlUltraclearultracentrifugetubes(Beckman#344058)

PreparationofRatLiverCytosol

1. Chillthesuperspeedcentrifuge,ultracentrifuge,SS-34and50.2Tirotorto4oC.
2. Inthecoldroom,rinsetheliverwellinPBS,thenrinseinfreshlypreparedHomogenizationbuffer.
3. Weightheliver,returntothecoldroom,andfinelymincethetissuewithscissors.Transferthemincedtissuetoaglasshomogenizer,add1volumeofhomogenizationbuffercontaining0.5mMMgGTP.Keepingthehomogenizerimmersedinslushyice,homogenizewithaTeflonpestleby6slowpassesat3,000rpm.
4. Transferthehomogenateto31.5mlcentrifugetubes,andremovecellulardebrisbycentrifugationat10,000xg(9000rpm)for10minat4oCinaSS-34rotor.
5. Inthecoldroom,collectthesupernatant,transferitto31.5mlultracentrifugetubes,pairthembyweight,andclarifythecytosolbycentrifugationat100,000xg(29,000rpm)for60minat4oCina50.2Tirotor.
6. Collecttheclarifiedcytosolicsupernatant,disburseinto50ulaliquots,andfreezebyimmersioninliquidnitrogen.Storeat-80oCuntiluse.

PreparationofRatLiverOrganelleFractions.

1. Performsteps1-4asin"PreparationofRatLiverCytosol"aboveexceptinstep2,homogenizethemincedliverinhomogenizationbuffercontaining0.5mMMgGTPand0.25Msucrose.
2. Inthecoldroom,collectthesupernatant(clarifiedhomogenate)andadd2.3Msucrosestocktomakethefinalconcentration1.25Msucrose.
3. SetupasucrosedensitystepgrADIentina25mlultraclearultracentrifugetube.Allsucrosesolutionsshouldbemadebydilutionofthe2.3Msucrosestocksolutionwithhomogenizationbuffercontaining0.5mMMgGTPandchilledto4oC.Adda2ml2.0Msucrosecushiontothebottomofthetube.Beingcarefulnottodisturbthecushion,layer12mlofclarifiedhomogenatecontaining1.25Msucroseonthe2.0MsucrosebyusingaPipetteandallowingthesolutiontoslowlyruninasteadystreamdownthesideofthecentrifugetube.Thenadda12mllayerof1.1Msucrose,andfinallyan8mllayerof0.25Msucrose.Discardanyexcesshomogenate.Pairthegradientbyweightwithabalancetube.
4. Separatetheorganellesbydensitybycentrifugingthegradientat100,000xg(28,000rpm)for3hat4oCinaSW-28rotor.
5. Inthecoldroom,withaPasteurpipettecarefullyharvesttheoff-whitebandofGolgimembraneatthe0.25M/1.1Msucroseinterfaceandtheoff-whitebandofER(ER)membraneatthe1.1M/1.25Msucroseinterfaceandplacetheminseparate31.5mlultracentrifugetubesonice.
6. PerformaBradfordassaytodetermineproteinconcentrationoftheGolgiandERmembranefractions.
7. Dilutebothmembranefractionswith3volumesof0.25Msucroseinhomogenizationbuffercontaining0.5mMGTP,andpelletthemembranesbycentrifugationat100,000xg(29,000rpm)for1hat4oCina50.2Tirotor.
8. Inthecoldroom,discardthesupernatantsandseparatelyresUSPendthedense,stickyERandGolgimembranepelletsbyextensivetriturationinPMbuffercontaining0.25Msucroseand0.5mMGTPtoachieveaproteinconcentrationof5mg/ml.
9. Disburseinto20ulaliquotsandfreezebyimmersioninliquidnitrogen.Storeat-80oCuntiluse.
10. DeterminetheproperdilutionofERofGolgimembranesforuseintheMT/Organellemotilityassay.Viewvariousdilutions(inPMbuffer)ofmembranesinsimpleperfusionchambersbyVE-DICmicroscopy.Notethedilutionrequiredsothat~20%oftheareaofthemicroscopicfieldiscoveredwithorganelles.ForsettingupthemembraneMTmotilityassay,youwillmakeneedastockthatis6xthedilutionjustdetermined,whichwillbecalled6XMEMBRANES.*=Aspublishedin...Waterman-Storer,C.M.(Inpress)Microtubule/organellemotilityassays.In:CurrentProtocolsinCellBIOLOGy,J.S.Bonifacino,M.Dasso,J.B.Harford,J.Lippincott-Schwartz,andK.M.Yamada,eds.JohnWiley,NY.

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