| Assay Method Information | |
| | Human CD38 Hydrolase Assay |
| Description: | The ability of test compounds to inhibit human CD38 hydrolase activity was measured in a fluorescence-based assay using non-physiological NAD+ substrate analogue 1,N6-etheno NAD+ (ε-NAD). Recombinant human CD38 (0.8 nM) was preincubated with test compounds in 384-well black microplates for 30 min at 25° C. in PBS (—Ca2+/Mg2+) containing 0.005% BSA (pH 7.4). CD38 hydrolase activity was initiated by addition of 4 μM ε-NAD, which yields the fluorescent product 1,N6-etheno ADP-ribose. Formation of fluorescent product was followed using ClarioStar Plus (BMG) microplate reader by reading fluorescence (excitation λ=300 nm; emission λ=410 nm) at two time points, one immediately after substrate addition (t=0) and one at 10 min (t=10). Data was analysed by subtracting the values detected at t=0 from t=10 to correct for variation in baseline fluorescence. Fluorescence values were converted to percent inhibition using the average of high signal (CD38 and ε-NAD) and low signal (CD38 and ε-NAD in the presence of a tool CD38 inhibitor) control wells. IC50 values were determined from a 10-point, half log concentration response curve with a four-parameter logistic equation. |
| Affinity data for this assay | |
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