FIG. 3.

Western blot analysis and ELISA of pilin. (A) Western blot analysis of pilin in whole-cell lysates. Whole-cell lysates were adjusted to contain equal amounts of protein, solubilized by boiling in SDS-polyacrylamide gel electrophoresis sample buffer, and separated by electrophoresis on an SDS–12% polyacrylamide gel by using a standard Tris-glycine buffer system. Western blots were developed with PAO1 anti-pilin polyclonal antiserum and an alkaline phosphatase-conjugated antibody as the developing antibody (Promega). (B) ELISA against whole cells. Bacterial cells were bound overnight to microtiter plate wells, followed by incubation with the PAO1 anti-pilin polyclonal antiserum. The developing antibody was an alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin. The A₄₁₀ was read with a Dynetech MR500 plate reader. Values are the means of results from three trials of five replicates each.