C12 nbd ceramide

The purified koji glycosylceramide (0.5 or 1.0 mg) was dissolved in reaction buffer (50 mM Tris-HCl buffer pH 7.5, 0.5 % w/v Triton X-100). Intestinal extract corresponding to 2 mg protein diluted twofold with pure water was added to the solution. The reaction solution was incubated at 37 °C for 18 h or 30 h. The reaction was stopped by boiling for 5 min, and then the solution was freeze-dried. The samples were dissolved in chloroform–methanol (2:1, v/v) and applied to TLC analysis. TLC was developed with chloroform–methanol–acetic acid–water (20:3.5:2.3:0.7, v/v). Detection was performed with 2 mg/mL orcinol in 70 % H₂SO₄ reagent. Ceramidase activity was measured using C12-NBD-ceramide (Avanti Polar Lipids, Inc., AL, USA) as the substrate (Mitsutake et al. 2001 (link)). C12-NBD-ceramide (1 nmol) was incubated at 37 °C for 18 h with 0.5 mg of the intestine extract. Chloroform–methanol (2:1, v/v) was added to the reaction mixture, the lower phase was collected and applied to a TLC analysis plate and developed with chloroform–methanol–25 % ammonia (90:20:0.5, v/v) and visualized by fluorescence.