Irdye conjugated anti mouse and anti rabbit secondary antibodies

Ab17978 overnight cultures were diluted in 10 mL fresh LB to an OD₆₀₀ of 0.05 in 50-mL conical tubes and grown at 37°C with shaking for 4 h until they reached an OD₆₀₀ of 1.0. For growth under TMP/SMX conditions, the concentrations of 3 μg/mL for TMP and 15 μg/mL of SMX were used for Ab17978, and TMP/SMX (1/4 μg/mL) was used for M2. For other antibiotics, bacteria were treated with antibiotic concentrations under which the final culture OD₆₀₀ values were between 65 and 85% of the untreated control. The cells were then pelleted by centrifugation and resuspended in Laemmli buffer to a final OD₆₀₀ of 20. Then, 5 μL of whole-cell samples was loaded onto 15% acrylamide SDS-PAGE gels for separation, transferred to a nitrocellulose membrane, and probed with polyclonal rabbit anti-CsuA/B (1:2,000) (42 (link)) and monoclonal mouse anti-RNA polymerase (anti-RNAP) (1:2,600) (BioLegend, San Diego, CA). Western blots were then probed with IRDye-conjugated anti-mouse and anti-rabbit secondary antibodies (both at 1:15,000) (LI-COR Biosciences, Lincoln, NE) and visualized with an Odyssey CLx imaging system (LI-COR Biosciences). Western blot signals were quantified using LI-COR Image Studio software, and the ratio of Csu to RNA polymerase intensity was calculated for at least 4 biological replicates.