Lipid Extraction and Separation by Thin Layer Chromatography (TLC)

Lipid extraction from yeast cells was performed using method described before (Dittrich-Domergue et al., 2014). Then, the total lipids were dissolved in chloroform and used for thin layer chromatography (TLC) to separate the neutral lipids on TLC silica Gel 60 plates. The solvent system used for development was hexane/diethyl ether/acetic acid (80:20:2, v/v/v). Lipid spots were revealed by spraying the plates with 0.01% primuline in acetone/H₂O (60:40; v/v) and visualized under ultraviolet light at 366 nm. TAG spots were scratched from the plate for making fatty acid methyl esters (FAMEs) and gas chromatography (GC) analysis. An appropriate amount of internal standard TAG 17:0 was mixed with samples loaded on the plate for TAG quantification.

Fatty acid methyl esters were derived using 2 mL of methanol that contained 2.5% sulfuric acid (v/v). The mixture was incubated at 80°C for 2 h, after which 2 mL of 0.9% NaCl (w/v) and 3 mL hexane were added. The mixture was homogenized and centrifuged to separate the layers. The upper organic phase was transferred into a new glass tube, and used for GC analysis as described in Yuan et al. (2015).

Total lipids were extracted from Arabidopsis seeds using chloroform/methanol (2:1, v/v), and resuspended in chloroform for subsequent methyl esterification, GC and TLC analyses as described previously. An appropriate amount of FA 17:0 was added as internal standard for quantification of total lipids.