Protean 2 xi cell electrophoresis system

One millilitre of culture of OD₆₂₀ = 0.6 was washed once with PBS and boiled 5 min in samples buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 2% β-mercaptoethanol, trace bromophenol blue). Whole cell lysates were analysed on Tris-glycine SDS-PAGE gel in a Protean II xi cell electrophoresis system (Bio-Rad) and visualized by Coomassie staining or transferred to nitrocellulose for Western blotting. Membranes were blocked with 5% BSA in PBS for 1 h, incubated 2 h with 1:2000 diluted α-OmpP5 rabbit polyclonal antibody with 0.5% BSA in PBS, washed five times for 5 min with Tris buffered saline + 0.05% Tween-20, incubated with 1:5000 diluted HRP-labelled donkey anti-rabbit Ig (GE Healthcare) and washed five times for 5 min with Tris buffered saline + 0.05% Tween-20. Binding was detected with ECL Western blotting substrate (Pierce) with a Fujifilm LAS-1000 scanner in an intelligent dark box II (Fuji Film). Densitometry analysis was performed with ImageJ (Schneider et al., 2012 (link)). Relative spot intensity was calculated by dividing the intensity of the OmpP5 protein as determined by Western blot by the intensity of all proteins in the Coomassie stain as loading control.