Nitro blue tetrazolium chloride 5 bromo 4 chloro 3 indolyl phosphate

SDS-PAGE and Western blotting procedures were done by standard techniques. Blots were developed with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Sigma–Aldrich) as a substrate, using previously obtained rabbit polyclonal anti-CjaA, anti-CjaD, anti-rCjaAD (Pawelec et al., 2000 (link); Łaniewski et al., 2012 (link)), or anti-LysM (this work) sera as primary antibodies, and mouse anti-rabbit IgG alkaline phosphatase conjugate (Sigma–Aldrich) as secondary antibodies.

Whole cell lysates were resuspended in 2× Laemmli Buffer (Sigma) and boiled for 5 minutes. Proteins were visualized by Western immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) on precast polyacrylamide protein gels (NuPAGE 4%‐12% Bis‐Tris/MES, Invitrogen), as described previously.16 Proteins were transferred onto nitrocellulose membrane using the iBLOT (Life Technologies). H pylori proteins were reacted with rabbit anti‐CagA serum 17 (1:2000 dilution). Secondary alkaline phosphatase‐conjugated goat anti‐rabbit antibodies and NBT/BCIP (Nitro blue tetrazolium chloride/5‐Bromo‐4‐chloro‐3‐indolylphosphate, Sigma) were used to detect antibody‐CagA complexes.

SDS-PAGE and Western blotting procedures were done by standard techniques. Blots were developed with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Sigma–Aldrich) as a substrate, using previously obtained rabbit polyclonal anti-CjaA, anti-CjaD (Pawelec et al., 2000 (link); Łaniewski et al., 2012 (link)), or anti-rCjaAD (this work) sera or anti-His antibodies (Sigma–Aldrich) as primary antibodies, and mouse anti-rabbit IgG alkaline phosphatase conjugate (Sigma–Aldrich) or goat anti-mouse IgG alkaline phosphatase conjugate as secondary antibodies.