In vitro deamidation of ubiquitin and native PAGE

Ten μg ubiquitin was incubated with 1 μg MvcA or MvcA mutants in 50 μl reaction in a buffer containing 50 mM Tris–HCl (pH 7.5) at 37°C for 2 h. 20 μl reaction mixtures were mixed with 5 μl 5× native gel loading buffer (pH 8.8) and loaded onto 10% native gels; gels were run in a buffer containing 375 mM Tris‐glycine (pH 8.8). Protein bands were visualized by Coomassie Brilliant Blue staining.