Sigmafast nbt bcip tablets

Each of the OMP preparations (5 μg) were run on the Novex Bis-Tris pre-cast gel system with MOPS running buffer according to the manufacturer's instructions (Life Technologies). Ammoniacal silver staining was carried out to visualize proteins. Western blotting was carried out using nitrocellulose membranes (Bio-Rad) and standard protocols68 . All mouse (AD6 anti-HMW1/2A34 (link); 1F4 anti-Hia35 (link)) and rabbit (LB1 anti-OMP P5 (ref. 28 (link)); chimV4 anti-OMP P5; anti-OMP P5) primary antibodies were used at a dilution of 1:2,500; all chinchilla (anti-OMP P2; anti-LPD27 (link); anti-PDM27 (link); anti-OMP P5/P6) primary antibodies at a dilution of 1:250. Anti-mouse-AP and anti-rabbit-AP secondary antibodies were used at a dilution of 1:5,000 (Sigma-Aldrich); protein A-AP secondary antibody was used at a dilution of 1:500 (Sigma-Aldrich) in blots where chinchilla primary antibodies were used. Blots were developed using SigmaFAST NBT/BCIP tablets according to the manufacturer's instructions (Sigma-Aldrich). All the primary antibodies were raised by the authors laboratories, with specific references for those described previously.

N. gonorrhoeae 1291 and clinical isolates grown under oxygen-limited conditions or N. gonorrhoeae 1291 aniA::kan cells grown under standard conditions were adjusted to an OD_600nm of ~2.5 in PBS for SDS-PAGE using NuPAGE 4–12% Bis-Tris polyacrylamide gels (Life Technologies). Pre-immune or post-immune sera were diluted appropriately in 5% skim milk powder in TBS-T as the primary antibody. Secondary antibodies used were anti-mouse IgG AP-conjugate or anti-rabbit IgG AP-conjugate (Sigma-Aldrich) at a 1: 10 000 in 5% skim milk powder in TBS-T. Antibody binding was detected with SigmaFAST NBT/BCIP tablets (Sigma-Aldrich) according to the manufacturer’s instructions.