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

Reference
Ralser M, Goehler H, Wanker EE, Lehrach H, Krobitsch S., Biotechniques. 2005 Aug;39(2):165-6, 168, PMID: 16116786