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An Effective Protein Extraction Method From Stationary Phase Yeast Cells

The stationary phase yeast cells are hard to break due to cell wall. This becomes even bigger problem when cells are treated with crosslinking reagents. A bead beater does a good job to breaking cells grown under any growth condition. An alternative method is grinding the yeast cells in liquid nitrogen, and boiling them in microwave.

Protocol

  1. Harvest the stationary phase yeast cells by either centrifugation or filtration through 45 microns nylon filter.
  2. Wash cells twice with ultrapure water.
  3. Weigh the cell pellet and transfer it into a mortar pre-chilled at -80°C.
  4. Add liquid nitrogen onto pellet and grind the cells into powder.
  5. Before the powder starts thawing, transfer it into a tube containing 1 volume of Protein Lysis buffer containing proteinase inhibitors and 1 volume 2x Tris-Glycine SDS Sample Buffer.

    Add 1 ml protein lysis buffer per 1 g of wet yeast pellet.

  6. Mix well. Place the tubes in water in a beaker and microwave until water starts boiling. When water starts boiling, continue microwaving another 5 minutes.

    7. It is important that the lysate volume is not more than 1/8 of the total volume of the tube, otherwise pressure will build up and cap will open up. You will lose the samples.

    It is important that you microwave the tubes. Keeping the tubes in a boiling water on the top of a heater does not efficiently break the unbroken stationary phase cells.

  7. Spin down the tubes at ~16000x G for 10 minutes in a table top centrifuge.
  8. Recover the supernatant containing soluble proteins, load 5 microliter on an SDS page gel to analyze.

    9. Before doing a western blot analysis, I suggest that you do a Coomassie Staining of the gel and check the efficiency of the lysis. It is always good idea to include exponential/log phase cells as a lysis control since they break up much more easily than stationary phase cells.

by Erbay Yigit