FIG. 4.

Soluble toxin-coregulated pilus protein A (TcpA) can interact with some anti-TcpA peptide antisera. (Top) A pulldown analysis which assessed the ability of anti-classical TcpA peptide serum to interact with TcpA monomers was performed. Purified, biotin-labeled TcpA was incubated overnight at 4°C with anti-TcpA peptide prebleed or tertiary serum, and then 20 μl of streptavidin-Sepharose (in PBS-1% nonfat milk) was added for 2 h at room temperature, followed by washing, electrophoresis, and subsequent Western blot analysis with horseradish peroxidase-labeled goat anti-rabbit IgG, antibody. (Bottom) Purified TcpA lysate was incubated overnight at 4°C with His-Select nickel affinity beads and washed, and anti-TcpA peptide preimmune (PI) or tertiary serum was added and incubated overnight at 4°C. Samples were washed before pellets were resuspended and analyzed in a Western blot as described for the top panel. rIg, rabbit IgG control.