Anti mouse antiserum conjugated to horseradish peroxidase

Proteins were separated by SDS-PAGE electrophoresis using 4–12% or 12% polyacrylamide NuPAGE Bis-Tris Precast Gels (Invitrogen). For SDS-PAGE analysis, gels were stained with SimplyBlue Safe Stain (Invitrogen). For Western blot (WB) analysis, proteins contained in the gels were transferred onto nitrocellulose membranes. Western blots were performed according to standard procedures. NHBA full-length protein and its C-terminal fragments were identified with polyclonal mouse antisera raised against the recombinant NHBA full-length protein (working dilution 1:1,000) or against the recombinant C2 fragment (working dilution 1:1,000), respectively. An anti-mouse antiserum conjugated to horseradish peroxidase (Dako) was used as secondary antibody. Bands were visualized with Super Signal West Pico Chemiluminescent Substrate (Pierce) following the manufacturer’s instructions. Densitometric analysis of protein bands was performed using Image J software.

To prepare total cell extract, N. meningitidis strains were grown overnight on GC agar plates at 37°C in 5% CO₂. Colonies from each strain were collected and used to inoculate GC broth to an initial optical density at 600nm (OD600) of 0.05–0.06. The culture was incubated at 37°C with shaking until an OD600 0.5 was reached, and then centrifuged for 5 minutes at 8000 × g. The supernatant was discarded and the pellet was resuspended in SDS-containing sample loading buffer. Total lysates were boiled for 5 minutes, separated by SDS-PAGE using the NuPAGE Gel System (Invitrogen) and transferred onto nitrocellulose membranes for Western blot analysis. Western blots were performed according to standard procedures. The different NHBA peptides were identified with polyclonal mouse antisera raised against the recombinant NHBA protein, corresponding to MC58 protein (peptide 3) (diluted 1:1000). Anti-PilE mouse polyclonal serum was used to determine the strain piliation status and a monoclonal anti-Opc antibody (B306) was used to evaluate Opc expression. In all cases, an anti-mouse antiserum conjugated to horseradish peroxidase (Dako) was used as the secondary antibody. Bands were visualized with Super Signal West Pico Chemiluminescent Substrate (Pierce) following the manufacturer’s instructions.