Pierce ecl chemiluminescent western blotting substrate

Stationary phase bacterial cultures in Luria–Bertani medium at an absorbance of 0.05 at 600 nm were grown at 30 °C for 16 h. Log phase cultures were grown overnight and diluted into fresh Luria–Bertani medium at an absorbance of 0.05 at 600 nm and grown to absorbance of 0.3 AU at 600 nm at 30 °C. Samples of 1 ml or 5 ml, respectively, were collected at 0, 30, 60, 120, and 180 min. Spectinomycin (Sigma) (200 μg ml⁻¹) was added at 0 min. Proteins were precipitated with 15% trichloroacetic acid (Sigma) for 30 min at 4 °C. Suspensions were then centrifuged at 5000 × g for 10 min at 4 °C. Pellets were isolated and washed with acetone for 10 min at 4 °C followed by centrifugation at 10,000 × g for 10 min at 4 °C. Acetone was removed, and pellets were resuspended in 10% SDS. Protein samples were analyzed by reducing SDS–PAGE and transferred to a nitrocellulose membrane (Invitrogen). Membranes were washed with Tris-buffered saline (pH 7.6) and Tween-20 (0.05%), blocked for 2 h with 2% (w/v) bovine serum albumin, probed with rabbit MinD polyclonal antibody serum and goat anti-rabbit IgG coupled with horseradish peroxidase. MinD was visualized using Pierce ECL chemiluminescent Western blotting substrate (ThermoFisher, Waltham, MA), and relative levels were quantified by densitometry using ImageJ (National Institutes of Health).