Gel bands excising and in-gel digestion
Protein bands visualized on 1D SDS-PAGE gel by Imperial Staining solution were excised and cut with a clean razor blade into 1×1 to 2×2 mm pieces. Gel pieces were placed into a 1.5 ml low-binding Eppendorf microcentrifuge tube and 200 µL of destaining buffer (40% Acetonitrile) was added. Samples were incubated at 37°C for 30 min with shaking. Destaining buffer was then discarded and gel pieces were washed with destaining buffer another time until the Coomassie dye was no longer visible in the gel. In-gel tryptic digestion was performed according to a previously published protocol (Paulo, 2016).