Deae sepharose cl 6b column

Clostridium difficile TcdA was purified according to Sullivan et al. [21 (link)]. In brief, a C. difficile strain 027 or 078 clinical isolate was inoculated with 50 ml BHI for 24 h and then transferred to dialysis bags (12–14 kDa exclusion, Fisher Scientific, USA) and grown for 72 h anaerobically in 1 l BHI. The contents were centrifuged to remove the bacteria, and the supernatant was filtered through a 0.22-μm filter. The culture supernatant was then concentrated using Centricon plus-70 membrane filters (>30 kDa, Millipore, Germany), loaded onto a DEAE-Sepharose CL-6B column (Sigma Aldrich, Canada) and equilibrated (50 mM Tris, pH 7.5), followed by elution with a gradient of 0.05–0.25 M NaCl at 1 ml/min. Fractions were assayed for bioactivity using a fibroblast cytotoxicity assay (described below), and presence of TcdA was verified by Western blot analysis (anti-TcdA antibody, 1:1000 dilution, Santa Cruz Biotechnology, USA). Fractions with TcdA were pooled, concentrated (Amicon ultra-15 filters, Millipore, Germany), aliquotted and stored at −80 °C.