SDS-PAGE and Western Blot Analysis

10% acrylamide gels were run at 100 volts for 100 minutes to resolve the purified samples along with a protein molecular weight marker (Bio-Rad, USA) and used for coomasive brilliant blue R-250 staining. Resolved proteins in SDS-PAGE gels were transferred to a nitrocellulose membrane and non-specific sites were blocked with 5% milk. In-house monoclonal antibodies against VP1 (mAb 51) [23 (link)], VP2 (mAb 7C7) [24 (link)] and polyclonal antibody against VP3 (raised against rVP3 from E.coli) were used as primary antibodies. Goat anti-mouse antibody (in 5% skim milk), conjugated with HRP, was used as secondary antibody. Detection was done using Enhanced Chemiluminescence (ECL) Plus western blotting detection reagent (GE Healthcare, UK).