2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis
Medium samples (10 µL) harvested from semi-continuous cultures were mixed with SDS sample buffer (5 µL) with or without 2-melcaptoethanol and boiled for 5 min. Sample solutions were electrophoresed on 4%–20% precast polyacrylamide gels (#4561094; BioRad, Hercules, CA, USA). Protein bands were visualized by incubating gels in protein staining solution (#LC6060, SimpleBlue SafeStain, Invitrogen) for 1 h. Next, gels were immersed in distilled water for 2 days to remove excess dye. Purified scFv-Fc was prepared from culture medium using a Protein A Sepharose column (rProtein A Sepharose Fast Flow, #17127901; Cytiva, Marlborough, MA, USA).[31]