Yeast Protein Extracts

by David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern

This protocol is a simple, reliable method for the preparation of yeast protein extracts for analysis by polyacrylamide gel electrophoresis (PAGE) and Western blotting. It works for both growing and stationary phase cells, grown either in liquid or on plates (YPD or minimal). A sample of 2.3 mg (wet weight) of cells yields sufficient protein to run several lanes on a minigel.

REAGENTS

• Appropriate yeast culture (liquid or plate)
• NaOH, 0.2 N
• PAGE sample buffer 2X

METHOD

1. Collect approximately 2.5 OD₆₀₀ of cells (~; 2.3 mg wet weight) from a liquid culture or scraped from the surface of a plate with a bacteriological loop. Resuspend the cells in 100 µl of H₂O, add 100 µl of 0.2 M NaOH, and incubate the sample for 5 minutes at room temperature.

2. Centrifuge the sample, and resuspend it in 50 µl of PAGE sample buffer. Boil the sample for 3 minutes and centrifuge again. Use a pipette to remove the supernatant, which contains protein.

3. Analyze 6 µl of the supernatant on a PAGE minigel.

4. If necessary, measure protein concentration in a diluted sample (1:3000) using the Bradford assay.