Anti mouse igg ap conjugate

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.

ES-62 was subjected to SDS-PAGE on 12% gels, and the gels were either stained with Coomassie Blue or transferred to PVDF membranes. Molecular weight standards were Precision Plus Kaleidoscope (Bio-Rad). Membranes were blocked with 1% (w/v) BSA in PBS containing 0.05% v/v Tween 20 and 0.5 mm CaCl₂ (PBSTC). Membranes were then incubated with 10 μg/ml CRP or TEPC15 (1 in 1000) in PBSTC or PBST containing 5 mm EDTA for 1 h at room temperature. CRP was detected with monoclonal anti-CRP (2C10) followed by anti-mouse IgG-AP conjugate (Sigma). Anti-PCh IgA binding (TEPC15) was detected with anti-mouse Ig-AP (Sigma). The chromogenic substrate was 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (in sodium carbonate buffer (pH 9.6) plus 2 mm MgCl₂). Quantitation of the relative density of protein staining in gels was performed using ImageJ.