Phospholipid Extraction磷脂提取

Hancock Laboratory Methods,Department of Microbiology and Immunology,
University of British Columbia, British Columbia, Canada http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=59 REAGENTS:

CHCl3:MeOH (2:1)
0.9% NaCl

METHOD:

Obtain 2 litres of cells in late log phase.

Centrifuge down and remove pellet to a glass container (corex tube) using a spatula.

Add 20 fold the pellet volume of CHCl3:MeOH (2:1) and agitate vigorously.

Centrifuge at 9000 x g for 20 minutes @ 4oC. Decant CHCl3 phase. It should be monophasic, but if it isn't, remove the bottom layer. (Avoid sucking up any precipitate or material floating on the lower phase.)

Add one fifth of the bottom layer volume of 0.9%NaCl. Mix vigorously. Recentrifuge as above. Discard upper phase and precipitate on interface.

Repeat step 5.

Reduce volume of CHCl3 to 1-2ml by evaporating under nitrogen. Phosphlipids can be quantitated by phosphate assay and identified by thin layer chromatography. Store at -20ºC or preferably -70ºC.