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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PURIFICATION: EXTRACT FROM CELLS
RAPID PREPARATION OF WHOLE CELL YEAST EXTRACT
Rapid Preparation of Whole Cell Yeast Extract
Contributor: The Laboratory of Jasper Rine at the University of California, Berkeley Procedure
1. Harvest cells from 50 ml of a log phase yeast culture (approximately 0.5 to 1.0 X 107 cells/ml) by centrifugation for 5 min in a clinical centrifuge at a setting of approximately "5".
2. Discard the supernatant and to the cell pellet add 500 μl of Extraction Buffer. Mix well by inversion and centrifuge at setting "5" for 1 min in a microcentrifuge.
3. Discard the supernatant and resuspend the cell pellet in 250 μl of Protease Buffer.
4. Add the cell suspension to a new microcentrifuge tube containing an equal volume of Acid Washed Glass Beads.
5. Vortex for 1 min.
6. Centrifuge in a microcentrifuge for 15 min at 15,000 X g (approximately maximum speed).
7. Save the supernatant and precipitate the cell wall debris by adding 0.2 volumes 100% Acetone (final
concentration 20% v/v).
8. Incubate on ice for 5 min.
9. Centrifuge in a microcentrifuge for 15 min at 15,000 X g (approximately maximum speed).
10. Save the supernatant and store at -70°C.
Extraction Buffer 15 mM MgCl2
20 mM EGTA Potassium Salt
50 mM HEPES Potassium Salt
80 mM β-Glycerol Phosphate
0.1% (v/v) Triton X-100
Acid Washed Glass Beads Wash extensively in ddH2O
Allow to cool at room temperature
425 to 600 μm Glass Beads
Soak in 1 M HCl for at least 2 hr
Bake to complete dryness
1 M HCl Protease Buffer 1X Pepstatin
0.5 mM PMSF (CAUTION! see Hint #1)
in Extraction Buffer
Chymostatin (100X) Store at -20°C
1 mg/ml Chymostatin
in DMSO (CAUTION! see Hint #1)
Pepstatin (100X) in Methanol
Store at -20°C
1 mg/ml Pepstatin
Leupeptin (100X) Store at -20°C
1 mg/ml Leupeptin
BioReagents and Chemicals
HEPES, Potassium Salt
EGTA, Potassium Salt
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.