Ecl light detection reagent

Cell extracts were separated on an 4/8 % SDS-PAGE gel and electro-transferred onto a PVDF membrane (Millipore Corp.) at 120 mA for 4 h. Membranes were blocked using 5 % BSA and 5 % non-fat dry milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl and 0.05 % Tween-20] overnight. Anti-BcsZ peptide antibody was used at 1:3000 dilution. Detection of CsgD was carried out using polyclonal anti-CsgD peptide antibody (1:5000) as the primary antibody [30 (link)]. Anti-OmpR and anti-DsbA antibodies were used as previously described. Goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories) conjugated with horseradish peroxidase at a 1:5000 or 1:2000 dilution, respectively, was the secondary antibody. FLAG primary antibody (Sigma) was used at 1:2000 dilution with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch) secondary antibody at 1:3000 dilution. After washing, binding of antibody was detected using the ECL light detection reagent (Roche). Visualization of bands was performed using FUJI LAS1000-plus chemiluminescence imaging system (Fuji, Stamford, CT, USA).

To detect CsgD expression, a 5-mg (wet weight) volume of bacterial cells from agar plates or liquid culture was placed into 200 µl SDS sample buffer and heated to 95°C for 10 min. The amount of protein was analyzed by Coomassie blue staining after gel separation. Sample volumes containing equal amounts of proteins were separated by SDS-PAGE (4% stacking and 12% resolving gel) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk overnight, and the protein was detected with a polyclonal E. coli anti-CsgD peptide antibody (1:5,000 dilution) (56 (link)). Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody (Jackson ImmunoResearch Laboratories Inc.) (1:2,000 dilution). The targeted proteins were visualized using ECL light detection reagent (Roche) and a Luminescent Image Analyzer (LAS-1000plus; Fujifilm).