Bligh-Dyer Method of Lipid Extraction

1) add 1.5 ml of CHCl3/MeOH (1:2) to a tissue sample in 0.4 ml of buffer and vortex vigorously CHCl3/MeOH/ H2O = 0.5:1: 0.4

2) add 0.5 ml of CHCl3 and vortex CHCl3/MeOH/ H2O = 1: 1: 0.4

3) add 0.5 ml of H2O and vortex CHCl3/MeOH/ H2O = 1: 1: 0.9

4) centrifuge briefly at 3000 rpm to separate phases (a white precipitate will appear at the interface)

5) transfer the organic (lower) layer to a new tube and concentrate by speed vac.

*If acidic lipids are to be extracted, add 0.5 ml of 1% HClO₄ instead of 0.5 ml of H₂O.

Also, the volumes in the above procedure may be modified but it is necessary to always keep the ratios of CHCl₃/MeOH/ H₂O as listed.