C12-NBD-ceramide

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
C12-NBD-ceramide is used as a fluorescent nCDase substrate and reaction products will be separated by reverse-phase HPLC^[2]:
1. 20 μM substrate, 1 nM nCDase in 75 mM NaCl, 25 mM HEPES (pH 8.0), and 0.4% Triton X-100, for 2 h at 37°C in a final volume of 100 μL.
2. Extract samples with 1:1 chloroform-methanol dries under nitrogen gas, and resuspends in 60 μL of HPLC mobile phase B. Separats reaction products by reversed-phase HPLC using a column(3 μm particle).
3. Mobile phase A contains 0.2% formic acid and 1 mM ammonium formate in HPLC-grade water. Mobile phase B contains 0.2% formic acid (IL) and 1 mM ammonium formate in HPLC-grade methanol.
4. FRET-based assay using 20 μM substrate ES.173.cds, 50 ng of nCDase, 75 mM NaCl, 15 mM sodium phosphate (pH 7.4), and 0.3% Triton X-100 for 3 h in 96 black well plates.
5. Measure resorufin fluorescence (Ex=347 nm, Em=430 and 530 nm).

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