TcdA and TcdB Binding Interaction Analysis

TcdA (1 μg) (List Biological Laboratories, Inc. California, USA) was incubated with DMC-4 (35 μl) for 1 h at 37 °C. Samples were loaded using 6X SDS loading buffer and heated at 95 °C for 10 min. All samples were loaded on to gradient SDS-PAGE gels (4-15%) after boiling. Silver staining was performed using the Pierce Silver Stain Kit as per the manufacturer’s instructions (Thermo Fisher Scientific. USA).
TcdA and TcdB (100 ng) were incubated with DMC-4 (35 μL) for 1 h at 37 °C. Samples were loaded using 6X SDS loading buffer and heated at 95 °C for 10 min. All samples were loaded into gradient SDS-PAGE gels (4-15%) after boiling. The polyvinylidene difluoride membrane (EMD Millipore, Germany) was incubated with either TcdA mouse monoclonal antibody (1:2500, Santa Cruz Biotechnology, Inc., USA, PCG4) or TcdB sheep polyclonal antibody (1:1,000 dilution, R&D Systems, USA, AF6246) overnight at 4 °C. After, the membrane was incubated with secondary antibody (1:20,000 dilution, goat anti-mouse HRP, Invitrogen, USA, CAT: 31430 for TcdA and 1:10,000, Donkey anti-Sheep HRP, Sigma-Aldrich, MO, USA, CAT: A3415 for TcdB) for 1 h at room temperature. Imaging of the membrane was performed using a Bio-Rad gel documentation system equipped with the Image Lab software^{26 (link)} (BioRad Laboratories Ltd., Canada).