Phospholipase c from b cereus

16:0–6:0 NBD-phosphatidylglycerol lipid (Avanti) was purified by preparative thin layer chromatography (TLC) as described previously ^{25 (link)}, dissolved in CH₃OH and stored at −20 °C until use. The lipid was dried and dispersed in octyl-glucoside by sonication prior to addition of the remaining components. Reaction mixtures contained 0.05 M sodium succinate pH 6.3, 10 mM MnCl₂, 0.05 M NaCl, 0.25 % octyl-glucoside, 20 μg NBD-phosphatidylglycerol, ultrasonically dispersed in 0.5 % octyl-glucoside (Branson 2200 bath sonicator) and either no enzyme, 20 μg eGacH, or 20 μg eGacH-T530A in a total volume of 0.02 mL. Following incubation at 37 °C for 3 hrs, the reaction was stopped by the addition of 0.08 mL CHCl₃/CH₃OH (2:1) and analyzed for fluorescence on a BioRad ChemiDoc MP Imaging System using the fluorescein preset mode, as described previously ^{25 (link)}. The migration position of the NBD-diacylglycerol product was determined from the product of a separate reaction containing purified phospholipase C from B. cereus (Sigma Aldrich).