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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.

Solutions
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
pH 7.5
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)
1X Chymostatin
in Extraction Buffer
1X Leupeptin
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
Leupeptin
Acetone
Hydrochloric Acid
DMSO
Glass Beads
Magnesium Chloride
HEPES, Potassium Salt
β-Glycerol Phosphate
EGTA, Potassium Salt
Methanol
Chymostatin
Pepstatin
Triton X-100
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.