Research Article

A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide

Figure 4

Substrate and time dependence of the kinase reaction. Recombinant bacterially expressed HsCERK was incubated with the indicated concentration of NBD-C6-Cer (a) or 5 μM (b) in the presence of 1 mM ATP at 37°C. The reaction was stopped at 10 min (a) or the indicated time periods (b) by addition of methanol and the mixture was applied to an NH2-SPE column. The reaction product NBD-C6-Cer-1-P was quantified based on fluorescence intensity of the column eluate and converted into nmol or μM, based on a calibration curve.
404513.fig.004a
(a)
404513.fig.004b
(b)