Western lightning chemiluminescence reagent kit

Toxins were partially purified and concentrated eight-fold from 72 hour C. difficile TY culture supernatants by methanol-chloroform precipitation [11 (link)]. Protein concentrations were determined using the BCA protein assay kit (Pierce) as per the manufacturer’s instructions. Concentrated supernatant proteins (10 µg) were separated by 10% (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [46 (link)] and transferred onto a nitrocellulose membrane (Whatman). Membranes were analysed as previously described [43 ]. TcdA and TcdB were detected using TcdA-specific monoclonal and TcdB-specific polyclonal antibodies (tgcBIOMICS), respectively. CDTa and CDTb were detected, respectively, using a CDTa-specific antibody and C. perfringens Ib-specific antibody that is cross reactive with CDTb [47 (link)]. CDTa, CDTb and TcdB-bound antibodies were detected using horseradish peroxidase conjugated anti-rabbit goat antibodies (Millipore) and TcdA-bound antibodies were detected using anti-mouse goat antibodies (Millipore). The Western Lightning Chemiluminescence reagent kit (Perkin-Elmer) was used to detect the bands, which were visualised by exposure to X-ray film or on a BioRad ChemiDoc XRS⁺ system.

Toxins were partially purified from culture supernatants by precipitation with 70% ammonium sulphate as described previously [6 (link)]. ADP ribosyltransferase assays were performed as previously described [48 (link)]. Briefly, precipitated supernatant protein (50 µg) was incubated for 60 minutes at 37°C with 10 µg of actin in assay buffer (20 mM Tris-HCl pH 7.5, 1 µM dithiothreitol (DTT), 40 µM ATP, 40 µM CaCl₂, 5 µM MgCl₂) and 10 µM of biotinylated NAD⁺ (Trevigen). The reaction was heat inactivated at 95°C for 5 minutes in 4x SDS sample buffer (240 mM Tris-Cl (pH 6.8), 40% glycerol (v/v), 8% SDS (w/v), 5% (v/v) 2-mercaptoethanol, 0.05% (v/v) bromophenol blue and separated by 10% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and biotinylated proteins were detected with horseradish peroxidase-conjugated streptavidin (GE Healthcare Life Sciences) and the Western Lightning Chemiluminescence reagent kit (Perkin-Elmer), following the manufacturer’s instructions. Relative band intensities were determined by densitometry using Image Lab Software (Bio-Rad). Data were analysed using GraphPad Prism 6 and statistical significance assessed using an unpaired t-test with a 95% confidence interval.