Preparation and Analysis of Cell Lysates

Cell lysates were prepared from exponentially growing cultures (OD₆₀₀ = 0.6–0.8) as previously described (Wan et al., 2013 (link)) with the following modifications. The equivalent of 1.0 ml of culture at an OD₆₀₀ of 0.6–0.8 was centrifuged at 16,000 × g for 5 min at 4°C. The supernatant was removed, and cell pellets were resuspended in 50 μl of 10mM Tris pH 8.0, followed by the addition of 50 μl of 2x SDS sample buffer. Samples were boiled for 5 min at 100°C before being run on a 12% (w/v) polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked for 30 min in 5% (w/v) non-fat dry milk in TBST (20 mM Tris, pH 8, 0.05% (w/v) Tween 20), and incubated at 4°C overnight with primary antibodies. Anti-FLAG tag and McpA antibodies were used at a concentration of 1:10,000. Then, a 1:10,000 dilution of secondary antibody, HRP-conjugated goat anti-rabbit immunoglobulin, was incubated with the membranes at room temperature for 2 h. Membranes were developed with SuperSignal West Dura Substrate (Thermo Scientific, Rockford, IL).