The Samuel Roberts Noble Foundation, Inc.   The Sumner Group: Protocols
 

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

Extraction Buffer (for 5 ml)

40 mM TRIS, pH 9.5  200 ul of 1 M
50 mM Mg Cl2    500 ul of 0.5 M
2 % PVPP (insoluble)  0.1 g
125 U/ml endonuclease    2 ul solution
1 mM PMSF     5 ul (dissolve 8.7 mg in 50 ul acetone)
Milli Q water
© 1997-2008 by The Samuel Roberts Noble Foundation, Inc.