| Assay Method Information | |
| | 17beta-HSD13 Biochemical Assay |
| Description: | More specifically, recombinant 17β-HSD13 protein was assayed in a buffer containing 200 mM Tris pH 7.5, 0.01% Triton X-100, and 0.02% BSA into a 384-well assay plate. Compounds were incubated with 17β-HSD13 (final 50 nM) and NAD+ (final 10 mM) at room temperature for 1 h prior to substrate addition. The assay reaction was then initiated by addition of β-estradiol (final 20 μM), and the reaction mixture was incubated for 2 hours at room temperature. Product formation was detected with chemiluminescence by adding equal volume of NAD+/NADH Glo reagent (Promega, #G9062) and read on a PHERAstar microplate reader (BMG LABTECH). IC50 values were determined with GraphPad Prism®, where log-transformed concentration values and the inhibition data were fitted to a four-parameter logistic equation. Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)). |
| Affinity data for this assay | |
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