Proteomics and Mass Spectrometry Facility, BRC, Cornell University Version 3.

2. TCA/Acetone Protein Precipitation Protocol

The following procedure is used to precipitate proteins from freshly prepared cell
lysates containing some contaminants, such as salt, detergents, nucleic acids, and lipids
that can interfere with the subsequent 2D analysis. TCA/Acetone Precipitation is one of
the most common precipitation methods. The acetone used in this procedure should be
pre-chilled and stored in a -20 °C freezer until needed and kept on ice during the entire
procedure.

Procedure

1. Mix the following solutions in a 1:8:1 ratio in the following order (invert tube after
adding each component):
1 ml cell lysate
8 ml 100% ice-cold acetone
1 ml 100% trichloroacetic acid (100%TCA, w/v)

2. Precipitate at -20 °C for 1 hr.

3. Centrifuge at 11,500 rpm (18,000 x g) for 15 min at 4 °C in a microcentrifuge.

4. Discard supernatant. Wash with 1 ml ice-cold acetone (resuspend pellet completely).

Centrifuge at 11,500 rpm for 15 min at 4 °C.

5. Repeat step 4 twice to remove all of the TCA.

6. Remove all acetone and let the pellet dry at room temperature. The presence of
residual acetone will make sample resuspension more difficult.

7. Store the pellet at -20 °C for late use.

8. Or dissolve the protein pellet in the appropriate volume of 2-D rehydration buffer
by repeatedly pipetting up and down to break up the pellet.

9. Allow sample to sit at room temperature for 1 hr, vortexing approximately every 10
min.

10. Transfer to Eppendorf tube and centrifuge at 14,000 rpm for 10 min at room
temperature.

Immediately transfer the supernatant into a new Eppendorf tube. Store at -80 °C until
ready for use. Discard pellet.