TCA/Acetone Precipitation (Large Scale)

AfCS Procedure Protocol PP00000148 Version 1, 05/12/03

The following procedure is used to precipitate protein from freshly prepared cell lysates. This allows for optimal protein recovery and accurate assays. The acetone used in this procedure should be prechilled 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) 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. Centrifuge at 11,500 rpm for 15 min at 4 C. 5. Discard supernatant. Dissolve the protein pellet in the appropriate volume of 2-D rehydration buffer 1 (2-D RB1) by repeatedly pipetting up and down to break up the pellet. 6. Allow sample to sit at room temperature for 1 hr, vortexing approximately every 10 min. 7. Transfer to Eppendorf tube and centrifuge at 14,000 rpm for 10 min at room temperature. 8. Immediately transfer the supernatant into a new Eppendorf tube. Store at -80 C until ready for use. Discard pellet. Reagents and Materials Acetone: Sigma-Aldrich; catalog no. A4206 Trichloroacetic acid, 100% (100% TCA); AfCS Solution Protocol PS00000507 2-D Rehydration buffer 1 (2-D RB1); AfCS Solution Protocol PS00000481

Author: Kathleen Lyons Date: 05/12/03 Approved: Deirdre Brekken