C12-NBD-ceramide,202850-01-9

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C12-NBD-ceramide

CAS No. : 202850-01-9

MCE 国际站：C12-NBD-ceramide

产品活性：C12-NBD-ceramide 是荧光神经酰胺类似物，可以作为底物用于神经酰胺酶实验中。

研究领域：Others

作用靶点：Fluorescent Dye

In Vitro: 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:
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).

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