Assayusesbutyryl-CoA(morehydrophobic) 14C-Cm+Bu-CoA->Bu-14C-CmKm(Cm)10µMKm(Ac-CoA)50µM The14C-ButyrylatedCmisseparatedfrom14CCmbyphaseextraction.ASSAY1.Makeupcocktail,keeponice;does10assays 0.25MTris-ClpH8.00.7ml(0.07/assay)14CCm(57µCi/µmol;25µCi/ml)0.1ml(10µl/assay)BuCoA(5mg/ml)0.2ml(20µM/assay) Total1.0ml(100µl/assay) forassayslongerthan1hr,mayneedtoaddextraBu-CoAtoreactionsat1hr. Reaction(setuponice) Extract(50µlof200total)50µlCocktail100µlTotal150µl Incubate37oC,30minto5hrtoovernight(1hrstandard) Tosamplesatroomtemp.add300µl(2vols.)xylene;vortexhard30secmicrocentrifuge3mins;remove270µl(care)add150µlTE,vortexhard,spin1min,removeallxyleneadd150µlTE,vortexhard,spin1min,remove250µlxylenetoascintillationvial.Add4mlscint,countuse 1CATunitacetylates1µmolchloramphenicol/min37oC
