Coomassie blue r250 staining

MMP-9 activation assay was performed as previously described with some modifications [10 (link), 14 (link)]. Briefly, 10 ng of recombinant human pro-enzyme MMP-9 (Calbiochem/EMD Millipore) were incubated with recombinant Zmp (100 µg), S. suis or S. pneumoniae (washed bacteria and supernatants as described above) for 1 h at 37 °C. Samples were then separated on 10% zymogram gels containing gelatin B (Sigma) under non-denaturing conditions. Results were visualized after Coomassie Blue R250 staining (Bio-Rad).

After native electrophoresis, the 120 kDa region of the gel was excised and eluted overnight at 4 °C in 1 mL of ProteoJet Mammalian Cell Lysis Reagent. For non-specific bound protein removal, the elution was incubated 1 h at 4 °C with a non-specific IgG and 100 μL of 50 % Protein A-Sepharose 4B beads (Invitrogen). After centrifugation, the supernatant was incubated at 4 °C with the antibody against eIF3f and 100 μL of 50 % Protein A-Sepharose 4B beads, washed 4 times with lysis buffer, resuspended in sample buffer (2 % SDS, 20 % glycerol, and 0.5 % bromophenol blue in 62 mM Tris HCl buffer, pH 6.8), boiled for 5 min, and subjected to electrophoresis on a 10 % SDS-polyacrylamide gel (10 % SDS-PAGE). A PageRuler Plus Prestained Protein Ladder (Thermo Scientific, Rockford, IL, USA) was included in all SDS-PAGEs. The resolved proteins were detected by Coomassie blue R250 staining (Bio-Rad); the unknown protein bands were excised and sent for N-terminal protein/peptide sequencing (Iowa State University of Science and Technology, Ames, IA, USA). After sequencing service, the partial amino acid sequences were subjected to a NCBI Blastp search to identify possible protein partners of eIF3f.