Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows: incubate 2 nmol of glycosphingolipid with 6 mU (1 μL) of EGCase I for 16 hours at 37°C in 50 mM sodium acetate buffer, pH 5.2 containing 0.1% Triton X-100. Total reaction volume is 20 μL. More complex substrates may require additional EGCase I for optimal results.