| Assay Method Information | |
| | Evaluation of HSD17B2 Activity Assay |
| Description: | Table 2: The fluorescence based detection assay monitors the conversion of the co-factor NAD+ to NADH, which occurs coincident with the conversion of estradiol to estrone by HSD17B2. These assays were performed in 384 well plates (Greiner V-shape PP-microplate). The 20 μl reaction volume contained: 0.7 μM estradiol (E2); 6 mM NAD+ (Sigma); 250 nM HSD17B2 enzyme (Origene; Cat #TP303293); 0.25 M potassium phosphate buffer pH 7.4, 0.75% vehicle (DMSO). Reactions were incubated for 2 hours at 37° C., and the reaction was stopped by freezing the reaction plates at −80° C. Zero time samples were frozen immediately.The conversion of NAD+ to NADH was quantitated using NAD-Glo kits (Promega; Cat #G9062) according to the manufacturers' instructions. A volume of 15 μL of enzyme reaction mixture was added to 15 μL of reconstituted Glo reagent, and the plates were incubated for 30 minutes at room temperature. Luminescence was quantitated on a Tecan Spark Reader®. A standard curve of NADH (0.1-50 μL) in potassium phosphate buffer pH 7.4/1% DMSO was run on each plate.Activity of the enzyme in the absence of E2 was used to evaluate specificity of conversion. Enzyme activity in the presence of test samples was expressed as a percentage of the uninhibited enzyme in the presence of 1% DMSO vehicle only, and was plotted versus inhibitor concentration. Non-linear regression was performed using a four-parameter logistic model and GraphPad Prism software. |
| Affinity data for this assay | |
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