Proti ace 2 kits

Cry14Ab was expressed in a plasmid-cured strain of Bt grown in liquid medium to sporulation and then pelleted. The spore/crystal pellets were extracted with 20 mM CAPS pH 10.5, 10 mM beta-mercaptoethanol, 10% glycerol, and the supernatant was run on a Superdex 200 gel filtration column in the same buffer. Fractions containing Cry14Ab were pooled and concentrated using Centricon centrifugal filter units with a molecular weight cutoff of 10 kDa. Purified protein at 0.4 mg/mL was incubated with 0.004 mg/mL of each protease (1:100 wt:wt ratio of protease to Cry14Ab) using Hampton Research Proti-Ace and Proti-Ace 2 kits, in 20 mM sodium acetate pH 5, 0.15 M NaCl, or 5 mM HEPES pH 7.5, 0.25 M NaCl, or 20 mM CAPS pH 10.4, 0.15 M NaCl, for 2 h at 37 °C. The reactions were halted by heating to 95 °C for 10 min in SDS–PAGE sample buffer with β-mercaptoethanol. 10 µL of each sample was run on SDS–PAGE and the gels were stained with Coomassie blue.

Twenty micrograms of purified proteins in their final buffer were added to 2.2 μg of eight proteases in 10 mM HEPES (pH 7.5), 500 mM NaCl, from the Proti‐Ace and Proti‐Ace 2 kits (Hampton Research), and incubated at 37°C for 1 h. The resulting products were then analyzed by SDS‐PAGE. As a control, we used 20 μg of purified proteins in 20 μl of enzyme buffer (10 mM HEPES (pH 7.5), 500 mM NaCl).