Western blotting was performed to determine the locations of the recombinant proteins. The whole cell proteins (WCP) and secretomes of M. bovis strains P1 and P150 were extracted as detailed above, separated by 12% SDS-PAGE, and transferred to PVDF membranes. After blocking, the signals were probed with mouse polyclonal antisera to specific recombinant proteins (diluted 1:200), and immunoblots were developed with HRP-conjugated goat anti-mouse IgG (1:3,000, SouthernBiotech, Birmingham, AL, USA) for 1 h.
Colony immunoblotting analysis was also performed to detect M. bovis cell surface or extracellular proteins, as described previously (25 (link)). M. bovis colonies grown on agar plates were transferred onto PVDF membranes through close contact. After blocking with 5% skim milk, the membranes were incubated with mouse antiserum (1:200) against each protein at RT for 2 h and then incubated with HRP-conjugated goat anti-mouse IgG antibodies. Both the western and colony blots were developed using an enhanced chemiluminescence substrate kit (Advansta, California, USA).
Colony immunoblotting analysis was also performed to detect M. bovis cell surface or extracellular proteins, as described previously (25 (link)). M. bovis colonies grown on agar plates were transferred onto PVDF membranes through close contact. After blocking with 5% skim milk, the membranes were incubated with mouse antiserum (1:200) against each protein at RT for 2 h and then incubated with HRP-conjugated goat anti-mouse IgG antibodies. Both the western and colony blots were developed using an enhanced chemiluminescence substrate kit (Advansta, California, USA).