2.5. SDS-PAGE Assay of Ab Proteolytic Activity

Analysis of Abs by SDS-PAGE was carried out in 4–15% gradient gels under nonreducing conditions (0.1% SDS), and the proteins were visualized by silver staining [15,16,25,26,35]. Before SDS-PAGE, IgGs (10–15 μg) were preincubated at 30 °C for 30 min in Tris-HCl (50 mM; pH 7.5), containing 1% SDS, and 10%. To restore the protease activity, SDS was removed by incubating the gel for 2 h at 22 °C with 4 M urea, and the gel was washed eleven times with water. Then 3–4 mm cross-sections of the gel longitudinal slices were cut out, incubated with 20 mM Tris-HCl (50 μL, pH 7.5), containing 5 mM MgCl₂ and 1 mM EDTA for four-five days at 4 °C to allow protein eluting from the gel and refolding. The eluates were used for the protease activity assay as described above. Parallel longitudinal slices of the gel were used to detect the position of IgGs in the gel by Coomassie staining.