Briefly, 20 μL of each sample was added to 5 μL reducing SDS PAGE sample buffer (Pierce, UK) and boiled for 5 minutes to denature the protein. Samples were then analysed by SDS PAGE using a 5% stacking gel and 15% resolving gel. After electrophoresis, gels were placed in a fixative solution (40% methanol, 15% acetic acid) and then stained with Brilliant Blue G (Sigma, UK). V8 protease samples were incubated on ice with 100 mM phenylmethanesulfonyl fluoride for 30 minutes prior to SDS PAGE in order to minimise self-digestion. The expected molecular masses of the V8 protease and α-haemolysin were given as 29 kDa and 33 kDa respectively, as specified
by the manufacturer. Statistical analysis Data are expressed as means ± standard error. The results of the azocasein hydrolysis assay and sphingomyelinase assay were analysed using www.selleckchem.com/products/Thiazovivin.html the univariate ANOVA test with Bonferroni ARRY-438162 mouse analysis. The results from the lethal photosensitisation of EMRSA-16 were analysed using the Mann Whitney U test. For both statistical analyses, a P value of less than 0.05 was considered statistically significant. For photosensitiser dose experiments, the P values refer to samples in the absence of light versus irradiated samples. For light dose experiments, the P values refer to samples in the absence of methylene blue
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