GlobalYeastExtractionProtocol

1) The yeast species Saccharomyces cerevisiae were cultured in SD media with 2% raffinose. Before extraction, yeast were precultured, diluted to 0.2 OD600 units/ml and cultured to 2 OD units/ml, washed and stored at -80oC.

 

2) Yeast cells were resuspended in 150 mM NH4HCO3  (pH 8) and disrupted by zirconia beads (0.5 mm; BioSpec Products).

 

3) Yeast cell lysates were diluted to 0.2 OD units (≈2*106 cells) per 200 μL, and mixed with 20μL of internal lipid standard mixture.

4) Samples were extracted with 990 μL chloroform/methonal (17:1 V:V) for 120 min. The lower organic 17:1 phase lipid extract was collected.

 

5) The remaing aqueous sample material was reextracted with 990μL chloroform/methonal (2:1 V:V) for 120 min. The lower organic 2:1 phase lipid extract was collected. Then the lipid extracts were vacuum evaporated.

 

6) The lipid extracts were dissolved in 100 μL chloroform/methonal (1:2 V:V).