Measurement of Protein Synthesis and Glycosylation

5. In intact cells, the incorporation of label into material comigrating with phosphatidylcholine is often great enough to overwhelm the signal from palmitoyl-CoA. If this is the case, aliquots of lipid extract can be enriched for palmitoyl-CoA by phase partitioning. Redissolve the dried lipid extracts in 1.1 ml chloroform/methanol/10 mM Tris, pH 7.4 (5 : 5 : 1, v/v/v), then add 0.5 ml chloroform and 0.275 ml water and split the phases as described above for the synthesis of [3H]palmitoyl-CoA. Transfer the phases and dry separately in a Speed-Vac concentrator. The upper aqueous phase will be substantially enriched in palmitoyl-CoA (see Fig. 2).