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Protocol: Chloroform/Methanol Precipitation

This method is a little tricky to perform but works well for precipitating proteins from many sources.

• Bring up predetermined amount of protein extract to 100 μl with water.
• Add 300 μl (3 volumes) water.
• Add 400 μl (4 volumes) methanol.
• Add 100 μl (1 volume) chloroform.
• Vortex vigorously and centrifuge; the protein precipitate should appear at the interface.
• Remove the water/methanol mix on the top of the interface, being careful not to disturb the interface.  Often the precipitated proteins do not make a visibly white interface, and care should be taken not to disturb the interface.
• Add another 400 μl methanol to wash the precipitate.
• Vortex vigorously and centrifuge; the protein precipitate should now pellet to the bottom of the tube.
• Remove the supernatant and briefly dry the pellets in vacuum centrifuge.
• Resuspend the pellets in a suitable amount of rehydration buffer.

Reference: Friedman, D.B. and Lilley K.S. “Quantitative proteomics for two-dimensional gels using difference gel electrophoresis (DIGE) technology” in John M Walker, Protein Protocols, 3rd Edition, Humana Press, 2009.