Quantifying RpoS Protein Levels in E. coli

Western blotting was performed as previously described10 (link). In brief, overnight cultures of the respective E. coli strains were diluted 1:100 using fresh LB medium (w/o antibiotics) and grown for 2.5 h at 37 °C with agitation (220 r.p.m.). The OD₆₀₀ of the cultures was monitored during bacterial growth and all samples were taken when the cultures had OD₆₀₀ readings between 0.69 and 0.79. For each sample, equal volumes of the liquid cell culture were pelleted by centrifugation (10,000g, 1 min) and the cell pellets were re-suspended in one-tenth of the original culture volume using a 1 × SDS loading buffer (80 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (w/v) ß-mercaptoethanol, 0.01% (w/v) brophenol blue). The samples were boiled for 5 min at 95 °C. Ten microlitres of each sample was loaded on a Tris-glycine SDS–polyacrylamide gel electrophoresis containing 10% (v/v) acrylamide. The primary antibody was the monoclonal anti-RpoS antibody (Neoclone, W0009) and used at a 1:1,000 dilution. The secondary antibody was an anti-mouse horseradish peroxidase-coupled antibody (GE Healthcare, NA931), which was diluted 1:5,000 in 1 × TBST with 4% (w/v) milk powder, prior to use. The protein bands were visualized using a homemade ECL reagent and standard film.