2.1.3. Thin Layer Chromatography Lipase Assay

PFL lipase activity against lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and various monoacylglycerols was assayed by incubating 0.5 µg PFL with 12.5 µg of lysophosphatidylcholine (Sigma-Aldrich, St Louis, MI, USA, 0.5 mM final concentration), lysophosphatidylethanolamine (Sigma-Aldrich), decanoyl glycerol (DG, Sigma-Aldrich, 1 mM final concentration), myristoyl glycerol (MG, Sigma-Aldrich, 0.8 mM final concentration) or oleoyl glycerol (OG, Sigma-Aldrich, 0.7 mM final concentration) in Hepes Buffer (Hepes 100 mM, pH 8) during 20 min at 50 °C, in a total reaction volume of 50 µL. At the end of the incubation, 50 µL of chloroform/methanol/acetic acid in a volumetric ratio of 50:50:1 (v/v/v) were added, and the sample was centrifugated for 1 min at 1000× g. Then, 8 µL of the organic phase were spotted on a silica gel 60W TLC plate, and the lipids were separated using a mixture of hexane-diethylether 60:40 (v/v) as eluent. Primuline, as a 0.5 mg/mL solution in an acetone–water 80:20 (v/v) mixture, was sprayed on the plate to stain the lipids, which were revealed by fluorescence on a Chemi Premium imager (blue illumination, 525 nm emission filter). Myristic and oleic acids were used as standards for the identification of the hydrolysis product.