Protocol: Precipitation with Trichloroacetic Acid in Acetone
A method often used for precipitating soluble proteins from plant tissues. Removes salts as well as hydrophilic and lipid soluble contaminants. However, precipitated proteins can be very difficult to redissolve, especially if the protein pellet is still quite acidic.
- Prepare a solution of 10% TCA, 20 mM DTT. Prepare a second solution of 20 mM DTT in acetone. Store both at -20°.
- Add 4 volumes -20° 10% TCA, 20 mM DTT in 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° 20 mM DTT in acetone. 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. Note: Bio-Rad recommends improving resolubilization of TCA/acetone precipitated proteins by pretreating the protein pellet with 20 µl 0.2 M NaOH prior to the addition of lysis buffer (500 µl), and incubating the protein pellet for 2 minutes.