Western Blot Analysis of Bacterial Proteins

Bacterial whole-cell lysate preparation and trichloroacetic acid precipitation of supernatant fractions from mid-exponential cultures was performed as previously described (24 (link), 104 (link)). Indicated fractions were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and proteins of interest were probed using polyclonal rabbit anti-His₆ (1:2,000; Invitrogen, Waltham, MA) and monoclonal mouse anti-RNAP (1:3,500; BioLegend, San Diego, CA). IRDye-conjugated anti-mouse IgG and anti-rabbit IgG were used as secondary antibodies (1:5,000 for each; LI-COR Biosciences, Lincoln, NE), and blots were visualized with the Odyssey CLx imaging system (LI-COR Biosciences).