Hyperfilm

NTCD strains were grown in BHI medium in an anaerobic chamber at 37°C for 24–48 h, after which culture supernatants and vegetative cell pellets were collected. C. difficile vegetative cell pellets were lysed in protein lysis buffer (dH₂O, 0.05 M Tris, 0.3 M NaCl, 0.5% TTX 100, 2 mM EDTA, 0.4 mM Na₃VO₄, 2.5 mM Leupeptin, 2.5 mM Aprotinin, 2.5 mM 4-Nitrophenyl 4-guanidinobenzoate hydrochloride [NPGB]). Protein concentration was measured using a BCA protein assay (Thermo Scientific, Suwanee, GA). Protein extracts were subjected to 12% SDS-PAGE separation. Then, proteins were transferred onto a Nylon membrane. After blocking for 1 h at room temperature with 5% skim milk, the membrane was incubated overnight at 4°C with anti-toxin A and anti-toxin B antibodies (1:1000). After washing with PBST (PBS with 0.05% Tween 20), the membrane was incubated with horseradish peroxidase-conjugated secondary goat anti-mouse antibody (Cat: ab97023, IgG, 1:3000, Abcam, Cambridge, MA), the antibody-reactive bands were revealed by enhanced chemiluminescence detection on Hyperfilm (Thermo Fisher Scientific, Waltham, MA).

Purified Tcd169 and Tcd169FI proteins were subjected to 8% SDS-PAGE separation. Then, proteins were transferred onto the Nylon membrane. After blocking for 1 h at room temperature with 5% skim milk, the membrane was incubated overnight at 4°C with anti-TcdA, anti-TcdB, or anti-sFliC antibody (Cat: 629701, Biolegend, Bath, UK). After washing with PBST (PBS with 0.05% Tween), the membrane was incubated with horseradish peroxidase-conjugated secondary goat anti-mouse antibody (Cat: ab97023, Abcam, Cambridge, MA), the antibody-reactive bands were revealed by enhanced chemiluminescence detection on Hyperfilm (Thermo Fisher Scientific, Waltham, MA).