Adapted from Yaffe,M.P.and Schatz,G.,PNAS,1984,81,4819-4823.

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 100μg/ml PMSF.

5.Add 150μl ice cold 2N NaOH 8% 2-ME (for 1 ml 400μl 5N NaOH 600μl ddH₂O 80μl 2-ME).Mix by inverting tube several times.Inc.on ice for 10 min.

6.Add 150μl 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 100μl sample buffer: 500μl 3X Sample buffer pH6.8 500μl ddH₂O 12.5μl 2-ME 25μl 1M TRIS base 100μg PMSF dab of bromphenol blue.You may have to work at this,I've heard a wire homogenizer does the trick.

9.Heat 95℃ x 2-5 min.Try loading 10μl on the gel,may need more.

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

•3X Sample Buffer:

•0.2 M Tris pH6.8

•6%SDS

•30% Glycerol