Protocol Acetone Precipitation

An excellent method for precipitating most soluble proteins. Removes salts and many lipid soluble contaminants and concentrates proteins. However, some of the precipitated proteins may not completely redissolve, particularly those that are very large or were originally membrane-bound.

  • Chill good quality acetone in a -20° freezer. Prepare a solution of acetone/water (4:1) and store at -20°.
  • Add 4 volumes -20° acetone to a sample extract. Vortex well. Place sample at -20° for 2 hours to overnight.
  • Spin at 13, 000 - 16,000 x g for 10 minutes at 0-4°. Carefully pour off the supernatant. Wash pellet twice with -20° acetone/water (4:1). During each washing, make sure the pellet is well broken up. Spin each washing at 13,000 - 16,000 x g, 0-4°, for 10 minutes.
  • Pour off final washing supernatant. SpeedVac samples for 10-15 minutes.
  • Dissolve in desired rehydration buffer or 25 µM ammonium acetate.