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