Anti mouse secondary antibody conjugated with hrp

Cells (strain LK134) were grown in LB rich medium to OD₆₀₀ 0.3 (time point 0). Subsequently, every 30 min 10 mL of cells were pelleted and OD₆₀₀ was measured. Pellets were washed with Lysis Buffer (20 mM Tris-HCl, pH 8, 150 mM KCl, 1 mM MgCl₂) and frozen. Next day, pellets were resuspended in Lysis Buffer (100–500 μL, according to the size of pellet) and disrupted by sonication 2 × 1 min, with 1 min pause on ice between the pulses. After centrifugation (5 min, 4 °C) to remove cell debris, the amounts of proteins were measured with the Bradford protein assay and 5 μg was resolved by SDS-PAGE and analyzed by Western blotting, using mouse monoclonal antibodies against the β subunit of RNAP (clone name 8RB13, dilution 1:1000, Genetex, Irvine, CA, USA) and anti-mouse secondary antibody conjugated with HRP (dilution 1:800,000, Sigma, Munich, Germany). Subsequently, the blot was incubated for 5 min with SuperSignal^TM West Femto PLUS Chemiluminiscent substrate (Thermo scientific, Waltham, MA, USA), exposed on film and developed.