1.Transfercellstoa15mltube.2.Add5mlTBS-toflasks,shake&pourintotubes.3.Spindownthecells@1krpmfor5".4.ResUSPendin1mlTBS-.5.Transferto1.5mltubes.6.Spindown@14KRPMfor2-3".7.Resuspendin100µl0.25MTrispH7.88.Freeze-thaw3x(10"freeze-10"thaw).9.Spindowndebris@14KRPMfor5".10.Transfer100µlsupernatanttoanewsetoftubes.11.Setnewsetoftubesandadd40µlofsupernatanttoeach.12.Inseparatetubemix:15µldH2O(timesnumberofsamples+1)70µl1MTrisPh8.0(timesnumberofsamples+1)20µl4mMAcetylCoA(timesnumberofsamples+1)and5µl14CCAM(timesnumberofsamples).13.Add0.5mlEthylAcetatetoeachtube.14.Vortex10"eachtube&thenmixbyinvertingforanother30".15.Spin@14KRPMfor30"-1".16.Remove450µloforganic(upper)phase.17.Speedvacfor15".18.Resuspendin30µlEthylAcetate.19.SpotonsilicagelTLC(in5µlamounts).20.DevelopTLCin200mlChloroform/Methyl-OH(95:5)untilsolutionisalmostatthetop(app.1hr.).21.Dry&exposeagainstfilmON.
