Trypsin proteolysis

To optimize the partial proteolysis condition, 2 μl of trypsin dissolved in water with variable stock concentrations was added into 18 μl pZIP4–ECD (0.15 mg/ml in 10 mM Tris–H₂SO₄, pH 7.0). After incubation at room temperature for 20 min, the reaction was stopped by adding 6 μl of SDS–PAGE loading buffer containing 1 mM PMSF. To test the effects of zinc ions on partial proteolysis, the reaction was performed in the presence of different concentrations of ZnSO₄ at the optimized protein:trypsin ratio (300 : 1, m/m). Partial proteolysis was analyzed by SDS–PAGE.