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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

YEAST MINI WHOLE CELL EXTRACT FOR WESTERN ANALYSIS USING GLASS BEADS

Yeast Mini Whole Cell Extract for Western Analysis Using Glass Beads
Contributor: The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
 
Overview
This protocol is an alternative to the Rapid Yeast Protein Preparation that involves boiling in SDS-PAGE buffer. It is suggested that this protocol be used if boiling the samples in SDS-PAGE buffer yields insufficient results.
 
Procedure
1. Grow a 100 ml yeast culture until the absorbance of the culture is about 1 at 600 nm (OD600 of 1). If the yeast culture becomes overgrown, use proportionally less cells for the preparation.

2. Pellet the cells by centrifugation at 5,000 X g for 15 min and remove the supernatant.

3. Resuspend the cell pellet in 20 ml of cold Extraction Buffer. Keep everything cold (on ice) from this point forward.

4. Centrifuge the cell suspension again and remove the supernatant.

5. Resuspend the cell pellet in 600 μl of Extraction Buffer and transfer the solution to 13 x 100 mm glass test tubes on ice. Add 400 μl of glass beads to each tube.

6. In a cold room, vortex the tubes for 1 min and incubate on ice for at least 1 min while vortexing the other tubes.

7. Repeat the vortexing step for each tube for a total of 5 cycles of vortexing and on-ice incubation.

8. Centrifuge the tubes in a clinical centrifuge for 3 min at its maximum speed.

9. Using a 1 ml pipettor, transfer the supernatant to a microcentrifuge tube, leaving most of the glass beads behind (transferring some of the yeast is permissible).

10. In a cold room, centrifuge the supernatant at maximum speed in a microcentrifuge. Transfer the supernatant to a fresh tube being careful not to transfer any of the pelleted cell debris.

11. Measure the protein concentration (See Protocol on determining Protein Concentration by a Microassay. Load 20 to 40 μg of protein on a protein gel for Western analysis (see Protocols on Westerns and SDS-PAGE).

Solutions
Chymostatin (2500X)   5 mg/ml in DMSO
Store at -20°C
Pepstatin (200X)   0.28 mg/ml in Methanol
Store at -20°C
Leupeptin (500X)   Store at -70°C
0.15 mg/ml in Ethanol
Benzamidine (100X)   Store at -20°C
31 mg/ml in ddH2O
PMSF (100X)   Store at -20°C
16 mg/ml in Ethanol
Extraction Buffer   1X Pepstatin
1X Leupeptin
1X PMSF
1X Chymostatin
1X Benzamidine
200 mM Tris, pH 8.0
2 mM DTT
1 mM EDTA
150 mM Ammonium Sulfate
10% (v/v) Glycerol
Add the DTT and protease inhibitors fresh on the day of the extract preparation
 
BioReagents and Chemicals
Benzamidine
Ammonium Sulfate
Ethanol
Tris
Methanol
EDTA
Glycerol
DTT
DMSO
Chymostatin
Pepstatin
Leupeptin
PMSF
Glass Beads
 
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