Glucosylation Assay for TcdB Activity

Glucosyltransferase (GT) activity of TcdB was measured by its ability to glucosylate Rho GTPase Rac1 in cell lysates (36 (link)). CT26 cell pellets were resuspended in a reaction buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 1 mM MnCl₂, and 2 mM MgCl₂), and lysed by passing through a 30 G needle for 40 times. After centrifugation (16,700 g, 3 min), the supernatant was used as a cytosolic fraction (protein concentration 2.5 mg/ml). To perform the glucosylation assay, the cytosolic fraction was incubated with TcdB at 10 ng/ml (with or without serum, sera were diluted at 1:200) at 37°C for 60 min. The reaction was terminated by adding SDS-sample buffer, and samples were heated at 100°C for 5 min before loading on a 12% SDS-PAGE gel. An antibody that specifically recognizes the non-glucosylated form of Rac1 (clone 102, BD Bioscience), anti-β-actin (clone AC-40, Sigma), and HRP-conjugated anti-mouse-IgG (Amersham Biosciences) were used for Western blotting.