Yeast Ethanol Lysates for SDS-PAGE and Western Blotting

Procedure

1. pick one colony

2. inoculate in 3 ml of the appropriate media

3. grow at 30° overnight

4. pellet the cells (5 min, 5000g)

5. wash 1X in sterile ddH2O

6. re- suspend the pellet in 200 µl EthOH (optional: +2 µl PMSF)

7. add approx. 100 µl glass beads (0.5 µ) in a reaction tube (you can use the cap of a 0.5 ml reaction tube as bucket)

8. vortex vigorously for 2 min., cold (optimal is an auto- vortex like “vortex turbo-mix”)

9. collect the supernatant in an fresh reaction tube

10. add again 200 µl EthOH

11. vortex

12. collect the supernatant

13. add again 200 µl EthOH

14. vortex for 2 min

15. (optional: repeat this again)

16. collect the supernatant

17. incubate at -20°C for 30 min or longer

18. centrifuge 16000g, for 15 min at 4°C

19. remove supernatant and re- suspend pellet in SDS page sample buffer and directly

20. boil the samples to denature

21. SDS Page and western blotting