2.6. Total Protein Extraction

Yeast cells were grown to mid-exponential phase, harvested by centrifugation and then stored at −80 °C before use. Cell pellets were thawed on ice and incubated in 2 vol of lysis buffer (1.85 M NaOH, 7.4% β-mercaptoethanol). To precipitate the proteins released from the lysed cells, a one-tenth volume of ice-cold 50% TCA was added. After incubation on ice for 20 min, the precipitated protein was collected by centrifugation at maximum rpm in a microfuge. After removal of the supernatant solution, the protein pellet was washed twice with chilled (−20 °C) acetone. To resuspend the precipitated protein and ensure neutralization of any residual TCA, the pellets were resuspended in 5% SDS in 0.1 M Tris base and then samples were diluted into SDS-PAGE sample buffer and analyzed by SDS-PAGE. For phosphatase treatment, TCA-precipitated proteins were solubilized in a solution constituted by mixing 42 μL of solubilization buffer (0.1 M NaCl, 0.3 M sorbitol, 25 mM MgCl₂, 1 mM EDTA, 10 mM Tris-Cl pH 7.5,) with 18 μL of 1 M Tris base and 40 μL of 10% SDS and 2% β-mercaptoethanol. The solubilized protein was then diluted with 900 μL of 50 mM Tris-Cl (pH 8.5), to which was added 90 units of calf intestinal phosphatase (New England Biolabs, Ipswich, MA, USA), and the resulting mixture incubated for 2 h at 30 °C in the absence or presence of phosphatase inhibitor (4 mM Na₃VO₄). Proteins were reprecipitated again, and then resuspended and resolved by SDS-PAGE, as above.