On-Cell Western Assay for Prototoxin Binding

To measure binding of prototoxin to MDCK.2 and ACHN cells the On-Cell Western assay was used as described previously [14] (link). Bound prototoxin was detected with mouse anti-Etx monoclonal Bio355 antibody (Bio-X Diagnostics S.P.R.L, Belgium) and IRDye 800CW goat anti-mouse IgG (H + L) antibody (LI-COR Biosciences, Lincoln, USA) at 1:500 dilution each. To quantify the amount of fluorescent signal, plates were imaged at 800 nm using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, USA). The binding activity of the mutant prototoxin was expressed as the percentage of fluorescence intensity relative to wild type prototoxin. To compare the means of the On-Cell Western assay data, Two-Way ANOVA analysis followed by Dunnett's multiple comparisons test was carried out using the GraphPad Prism 6 software (GraphPad Software, La Jolla).