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Yeast Ethanol Lysates for SDS-PAGE and Western Blotting

Source: Markus Ralser
Date Added: Mon Feb 02 2009
Date Modified: Mon Feb 02 2009
Abstract: This protocol describes a quick method for preparing yeast ethanol lysates for SDS page and western blotting

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 l EthOH (optional: +2 l PMSF)
  7. add approx. 100 l glass beads (0.5 ) 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 l EthOH
  11. vortex
  12. collect the supernatant
  13. add again 200 l EthOH
  14. vortex for 2 min
  15. (optional: repeat this again)
  16. collect the supernatant
  17. incubate at -20C for 30 min or longer
  18. centrifuge 16000g, for 15 min at 4C
  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

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