In vitro autoprocessing of TcdB toxin

The in vitro autoprocessing activity of TcdBs was performed in a 20 uL reaction with 200 nM of toxin, cleavage buffer (150 mM NaCl, 20 mM Tris pH 7.4), and 10 mM InsP6 (Sigma-Aldrich, St. Louis, MO, USA) [23 (link)]. The reaction was incubated for 0, 2.5, 5, 7.5, 10, 20, 30, 60, and 90 min at 37 °C. The reaction was stopped by addition of loading buffer and boiling at 95 °C for 5 min. The samples were separated by 7.5% SDS-PAGE and then analyzed by Coomassie staining. The activity of each toxin was quantified by comparing the densities of the bands of full-length and processed protein using Image J software (NIH, Bethesda, MD, USA). The data were plotted using nonlinear regression and in GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA [29 (link)].