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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.


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 Fl├╝gge, U.I. (1984) Anal. Biochem. 138 141–143.