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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

GLASS BEAD LYSIS OF YEAST

Glass Bead Lysis of Yeast
Contributor: The Laboratory of R. Daniel Gietz at the University of Manitoba.
URL: Lab Web Site
 
Procedure
1. Grow 25 ml of yeast cells to mid-log phase (approximately 1 x 107) (See Protocol on Growth of Yeast Cells).

2. In 50 ml conical tubes, pellet the cells at 2000 X g for 5 minutes in a centrifuge.

3. Discard supernatant.

4. Resuspend the cells in approximately 25 ml of ddH2O.

5. Centrifuge the cells again and discard the supernatant.

6. Resuspend the cell pellet in 1 ml of ddH2O and transfer the cell suspension to a 1.5 ml microcentrifuge tube.

7. Centrifuge the cells for 5 seconds at maximum speed in a microcentrifuge and discard the supernatant.

8. Resuspend the cell pellet in 0.5 ml of SDS-PAGE Sample Buffer plus freshly added PMSF to a final concentration of 0.5 mM and Benzmidine to a final concentration of 0.5 mM at 4°C.

9. Add approximately an equal volume (0.5 ml) of acid washed 0.45 μm glass beads.

10. Cap the tube tightly and vortex vigorously four times for 45 seconds each, with 30 second pauses on ice in between mixings.

11. Centrifuge the tubes for 5 minutes at maximum speed in a microcentrifuge at 4°C.

12. Transfer the supernatant to a new 1.5 ml microcentrifuge tube, cap the tube and boil the collected supernatant for 5 minutes.

13. Analyze 10 to 15 μl by SDS-PAGE (see protocol on SDS-PAGE).

Solutions
0.5 M Benzamidine
0.1 M PMSF   CAUTION See Hint #1
Prepare in Isopropanol
Store at -20°C
SDS-PAGE Sample Buffer   0.0025% (w/v) Bromophenol Blue
60 mM Tris, pH 6.8
5% (v/v) 2-mercaptoethanol
2% (w/v) SDS
10% (v/v) Glycerol
 
BioReagents and Chemicals
SDS
Benzamidine
2-Mercaptoethanol
2-Propanol
Glycerol
Bromophenol Blue
Tris
PMSF
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.