Precipitating Proteins

TCA precipitation

• 1. Add TCA 100% to the sample to reach the final concentration of 10%

  2. Mix by inverting the tube

  3. Incubate at 4°C for 30min or O/N

  4. Centrifuge for 15 min at 17000xg 4°C

  5. Discard supernatant

  6. Wash with 500 ul acetone 80% cold

  7. Centrifuge for 5 min at 17000xg 4°C

  8. Discard supernatant

  9. Dry pellets

  10. Resuspend in Loading Buffer

  11. equilibrate pH with Tris 1M pH 8 if needed

Acetone-precipitation

Dissolve 20ml of sample in 180l acetone. Do not forget to vortex! Let incubate at -20°C for at least 1 h. Spin at maximum speed and dry the pellet in the speedvac.

Optionally, add three drops of glacial acetic acid per 180 mlof acetone. Wash the pellet with pure acetone, redissolve in a proper volume of water.

Ammonium sulfate precipitation

Dissolve 20 ml of sample in 180 ml of saturated ammoniumsulfate. Icubate on ice for 30 min. Spin for 15 min at 14000 /min. discard the supernatant and redissolve in 20 ml water.