玻璃珠裂解酵母细胞方法

玻璃珠裂解酵母细胞方法

Glass bead lysis of whole yeast cells 

The following protocol is suitable for preparation of 5 OD600 units of whole-cell extract at a final concentration of 0.5 OD600 units per 20 µL of SDS-PAGE reducing sample buffer.

1. Dilute an overnight culture of yeast cells to 0.2 ODU/mL in 10 mL medium.

2. When the culture reaches mid-logarithmic stage (0.5 – 0.8 ODU/mL), centrifuge 5 ODU of cells at 500 x g for 5 min at room temperature (RT).

3. Decant supernatant, resuspend cell pellet in 1 mL sterile water, and transfer entire volume to eppi tube.

4. Add 100 µL of 100% trichloroacetic acid (TCA) and vortex immediately.

5. Incubate on ice ≥ 20 min. • If the day is late, place a lid over the ice bucket and leave the bucket in the cold room until the next morning.

6. Centrifuge at 14,000 x g for 5 min at 4˚C.

7. Aspirate supernatant; keep pellets on ice.

8. Add 1 mL ice-cold acetone. • Large volumes of acetone can be stored at -20˚C.

9. Resuspend pellet completely using a water bath sonicator.

10. Repeat steps 5 – 9.

11. Incubate on ice ≥ 20 min.

12. Centrifuge at 14,000 x g for 5 min at 4˚C.

13. Aspirate supernatant.

14. Dry pellets completely by speed-vac for 10-15 min. • Pellets can also be dried by leaving the tubes with the lids open at RT.

15. Add 200 µL of 1X SDS-PAGE sample buffer + 0.2% β-mercaptoethanol • 1X SDS-PAGE buffer: (remember to add β-mercaptoethanol to a final concentration of 0.2%).

16. Add 100 µL of sterile glass beads.

17. Secure eppi’s with lid-locks.

18. Vortex 15 min at RT.

19. Heat at 90 – 100˚C for 5 min.

20. Repeat steps 18 – 19.

21. Centrifuge 15 – 30 seconds at 14,000 x g at RT.

22. Load 20 µL (equivalent to 0.5 OD600 units) onto an SDS-polyacrylamide gel