Post-stained western blotting, a useful approach in immunoproteomic studies

• Exact localization of immunogenic proteins on stained 2DE-gels can be difficult.
• The suitability of post-stained western blotting to identify immunogenic proteins is evaluated.
• It allows an easily and accurate selection of immunogenic proteins before identification.

The precise localisation of immunogenic proteins on stained two-dimensional electrophoresis (2DE) gels is occasionally difficult, contributing to the erroneous identification of unrelated non-immunogenic proteins, which is expensive and time consuming. This inconvenience can be solved by performing immunoblotting using previously stained polyacrylamide gels. This approach was proposed nearly 20 years ago but is now almost forgotten. We have evaluated the suitability of this approach to identify immunogenic proteins from Lactococcus garvieae. Some of the immunogenic proteins identified in L. garvieae, such as Gls24, have been considered important as immunotarget in different bacterial species. Post-staining western blotting facilitated the correct selection of immunogenic proteins of interest in 2D gels before their identification.