N-[12-[(7-NITRO-2-1,3-BENZOXADIAZOL-4-YL)AMINO]DODECANOYL]-CERAMIDE

C12-NBD-ceramide is a fluorescent analogue of ceramide, it can be used as a substrate in ceramidase assays[1][2].

Research Areas >> Others

Signaling Pathways >> Others >> Others

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). In this experiment, 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 reverse-phase HPLC using a Spectra 3 µm C8SR column (3 μm particle, 3.0 × 150 mm). 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).

[1]. Tani M, et al. Specific and sensitive assay for alkaline and neutral ceramidases involving C12-NBD-ceramide. J Biochem. 1999 Apr;125(4):746-9.

[2]. Otsuka Y, et al. Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening. SLAS Discov. 2021 Jan;26(1):113-121.