C12 NBD Galactosylceramide

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Experimental steps (To determine whether acid ceramidase can directly cleave galactosylceramide)^[1]:
1. 20 μmol of C12-NBD-Galactosylceramide are incubated with 5 to 10 μg of purified acid ceramidase in a 30 μL reaction containing 15 μL of 0.2 M citrate phosphate buffer (pH 4.5), 2.25 μL of 2 M NaCl, 1.5 μL of 10 mg/mL bovine serum albumin (BSA), and 0.3 μL of 10% IGEPAL CA630.
2. Incubate at 37°C for 18 h without agitation and then stop by adding 60 μL of acidified methanol.
3. The amount of psychosine formed by the deacylase activity of the enzyme is determined by monitoring the release of NBD-fatty acid on an Acquity UPLC (excitation, 435 nm; emission, 525 nm).
4. 26 μmol of C12-NBD-galactosylceramides is incubated at 37°C with 1.0 μg of enzyme for 3 h or 24 h. Detected minute levels of psychosine to compare the efficiency of ACDase substrates.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.