Western Blot Protein Analysis Protocol

For SDS-PAGE analysis, cell extracts were loaded onto SDS-polyacrylamide gels, migrated, and transferred to a nitrocellulose membrane at 3 mA/cm². After transfer, membranes were blocked overnight in blocking buffer (5% milk powder, 0.1% Tween 20 in Tris-buffered saline, pH 8.0). Polyclonal antibodies against VgrG1a were used at a dilution of 1:1000 (22 (link)). Monoclonal anti-V5 antibody (Invitrogen) was used at a dilution of 1:5000. Monoclonal antibodies were used against the β subunit of RNA polymerase (NeoClone) 1:1000. Antibodies against PhoE and SecA were kindly provided by Jan Tommassen (Utrecht University) and used at a dilution of 1:2000 and 1:1000, respectively. Secondary antibodies conjugated to horseradish peroxidase were used at a dilution of 1:5000. Western blots were developed using Super-Signal West Pico Chemiluminescent Substrate (Pierce) and visualized on a LAS3000 Fuji Imager.