TRIS Extraction of Cells

1. Grind 3 g cells in liquid N₂ (can use only 1-2 g cells with an equivalent decrease in buffer volume)

2. Add 2 ml ice cold buffer - see recipe below

3. Vortex; probe sonicate x 3-4 times in short bursts (keep the sample cold)

4. Centrifuge for 10', 4 C, 12-14000 x g

5. Recover the supernatant. I usually do a quick protein check here (Bio-Rad/Bradford)

6. Precipitate on ice with an equal volume of 25% TCA for 5-10' or @ -20 with an equal volume of 10% TCA/acetone for > 1 hr

7. Centrifuge for 5-10' @ 12-14000 x g

8. Decant the supernatant and wash the pellet x 2 with acetone (200 ul, 3-5' spin, 12-14000 x g)

9. Air dry the pellet (approximately 10')

10. Resuspend in 8 M Urea, 4 % CHAPS, 20 mM DTT, 0.2-0.5% biolytes – biolyte concentration depends on whether you plan to use Bio-Rad or Amersham strips