Drummond:ChlorMethExtraction

Chloroform-methanol extraction of proteins

Detergents, like SDS, and salts, like NaCl, can disrupt LC-MS/MS runs. Precipitation with chloroform and methanol results in dry protein material, free of salt and detergent.

Protocol

To 100uL protein sample (~100ug protein) in a 1.5mL eppendorf tube:

• Add 400 uL methanol and vortex thoroughly.
• Add 100 uL chloroform and vortex.
• Add 300 uL H₂O—mixture will become cloudy with precipitate—and vortex.
• Centrifuge 1 minute @ 14,000g. Result is three layers: a large aqueous layer on top, a circular flake of protein in the interphase, and a smaller chloroform layer at the bottom.
• Remove top aqueous layer carefully, trying not to disturb the protein flake.
• Add 400 uL methanol and vortex.
• Centrifuge 5 minutes @ 20,000g, which will slam dandruffy precipitate against the tube wall.
• Remove as much methanol as possible. Be careful, because the pellet is delicate. You should be able to remove all but a few uL of methanol with care, which will speed drying.
• Dry under vacuum.

Adapted from Wessel, D. and Flügge, U.I. (1984) Anal. Biochem. 138 141–143.