Non radioactive fatty acids

Acyl-CoAs ([1-¹⁴ C]-labeled an unlabeled) used for enzymatic assays were synthesized according to the method developed by Sánchez et al. [47 (link)]. [1-¹⁴C]-fatty acids and triolein [carboxyl-¹⁴C] were purchased from Perkin Elmer or American Radiochemicals. Labeled diacylglycerol was derived through partial lipase treatment of triolein [carboxyl-¹⁴C] with Rhizopus arrhizus lipase (Sigma-Aldrich). The lipase products were separated on TLC plates (silica gel 60; Sigma-Aldrich). The DAG was eluted from the silica with methanol: chloroform (2:1, v/v), extracted with chloroform by Bligh and Dyer method (1959). Non-radioactive fatty acids and diacylglycerol were ordered from Larodan or Sigma-Aldrich. Internal standard of heptadecanoic acid methyl ester, coenzyme A and TAG standard for thin-layer chromatography were purchased also from Sigma-Aldrich.

[1-¹⁴C]-labelled fatty acids were purchased from Amersham Biosciences (Amersham, UK), and non-radioactive fatty acids and non-radioactive lipid standards from Larodan (Malmö, Sweden). Free CoA, bovine serum albumin (BSA), NADH and heptadecanoic acid methyl ester (17:0-Me) were supplied by Sigma-Aldrich (St. Louis, MO, USA). The [1-¹⁴C]-labelled acyl-CoAs were prepared according to the modified methods described by Sanchez et al. [32 (link)]. The other chemicals and solvents used for analysis were from Merck (Darmstadt, Germany) or Sigma-Aldrich.