Protein Extraction and Western Blotting

2 x 10⁷ log-phase cells were resuspended in 100 μl H₂O. An equal volume of 0.2 M NaOH was added to the cell suspension, and cells were incubated 5 min at room temperature. Samples were then centrifuged at 20,000 x g for 10 min at 4°C. Pellets were resuspended in SDS-lysis buffer (10 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1% SDS, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (leupeptin, pepstatin, PMSF, and aprotinin) for western blot analysis. Immunoblotting was carried out exactly as previously described (Hughes and Gottschling, 2012 (link)). Anti-GFP primary antibody was from Roche (Switzerland) (#11814460001), and secondary HRP-conjugated antibodies from Jackson Immunoresearch (West Grove, PA).