λ protein phosphatase

Yeast total protein extracts were prepared by vigorous shaking of cells in the presence of glass beads in a non-denaturing lysis buffer (150 mM KCl, 50 mM Tris-HCl pH 8.0, 20% glycerol, protease inhibitors cocktail from MERCK Millipore). The dephosphorylation assays were carried out by incubating 10 mg of cell extract in λ protein phosphatase buffer (50 mM Tris-HCl pH 7.8, 5 mM DTT, 2 mM MnCl2, 100 μg/ml bovine serum albumin) containing 200 U of λ protein phosphatase (MERCK Millipore) for 20 min at 30°C. Phosphatase reactions were stopped by adding an equal volume of 2x western sample buffer and incubating at 95°C for 2 min.