1. Grow 25mls yeast cells to 5x10E6.
2. Sequentially spin down cells in a 15 ml polypro tube.
3. Wash cells with 1ml ice cold ddH2O, transferrring to an eppy tube.
4. Recon. cells in 1 ml ice cold ddH2O containing 100ug/ml PMSF.
5. Add 150ul ice cold 2N NaOH + 8% 2-ME (for 1 ml 400ul 5N NaOH + 600ul ddH2O + 80ul 2-ME). Mix by inverting tube several times. Inc. on ice for 10 min.
6. Add 150ul ice cold 50% TCA, mix by inverting tube several times. Inc. on ice for 10 min.
7. Spin 2 min. in microfuge. Wash pellet with 1ml. ice cold acetone, spin 2 min. and aspirate off the supernatant. Dry pellet.
8. Resuspend pellet in 100 ul sample buffer: 500ul 3X Sample buffer pH6.8 + 500ul ddH2O + 12.5ul 2-ME + 25ul 1M TRIS base + 100ug PMSF + dab of bromphenol blue. You may have to work at this, I've heard a wire homogenizer does the trick.
9. Heat 95¡C x 2-5 min. Try loading 10ul on the gel, may need more.

Note: Make PMSF at 10mg/ml in isopropanol and store at -20¡C.

• 3X Sample Buffer:
• 0.2 M Tris pH6.8
• 6%SDS
• 30% Glycerol