Multiclonal Antibody Generation and Immunoblotting

PsaA, PiuA, and PiaA antibodies were derived from the previous work (Yang et al., 2016 (link)). The purified SPD_0090 protein was used as an antigen for the immunization experiments to generate multiclonal antibodies in mice according to the previous report (Brown et al., 2001b (link)). Equal amounts of proteins were analyzed by 10–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, United States), which were incubated with the corresponding antibodies at 4°C. This was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse as a secondary antibody. Results were visualized with Clarity Western ECL substrate (Bio-Rad, United States) and captured using Image Master 2D Platinum 6.0 (GE Healthcare, United States).