Antibodies against TcdC were generated by immunizing rabbits with a synthetic peptide (CQLARTPDDYKYKKV) representing a specific TcdC epitope (Genscript). Note that this epitope is located before the Clostron insertion site, and would therefore also be expected to detect truncated TcdC protein, would this be produced. Western blots were performed as follows. C. difficile (2 mL) cultures were harvested by centrifugation (2 min, 11.0006g, 4uC) and washed with Phosphate Buffered Saline (PBS). The bacterial pellets were resuspended in PBS containing protease inhibitor cocktail (Complete, Roche) and lysed by sonification. The bacterial lysates were centrifuged at low speed (3 min, 10006g, 4uC) to remove unbroken bacterial cells [20] . To separate the cytosolic proteins from the membrane associated proteins the bacterial supernatant was centrifuged at 200.0006g, 4uC for 1 hr [20] . The pelleted membrane associated proteins were resuspended in 10 mM Tris-HCl (pH 7.4), 5 mM EDTA with 2% Triton X-100 for 30 min at room temperature. Equal amounts of the resuspended membrane associated proteins were separated on 15% SDS PAGE gel and transferred onto polyvinyl difluoride (PVDF) membranes. Similarly generated membranes with the transferred membrane associated proteins of a Type 035 (PaLoc negative) strain were used for pre incubation of the TcdC antibodies. The membranes were probed with the pre incubated TcdC antibody and an antibody against the b subunit of the E. coli F 0 F 1 ATPase that cross reacts with the homologous protein in C. difficile [20, 43] . The probed membranes were analyzed using secondary anti-mouse horse radish peroxidase conjugated antibodies (Dako), a chemiluminescence detection kit (Amersham) and a Typhoon 9410 scanner (GE Healthcare).