Detergents, like SDS, and salts, like NaCl, can disrupt liquid chromatography/tandem mass spectrometry (LC-MS/MS) studies by interfering with chemistry and clogging columns and spray needles. Precipitation with chloroform and methanol results in dry protein material, free of salt and detergent, which perform sweetly during these critical steps.
To 100 µL protein sample (~100µg protein) in a 1.5mL eppendorf tube:
- Add 400 µL methanol and vortex thoroughly.
- Add 100 µL chloroform and vortex.
- Add 300 µL 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 µL 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 µL 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.