| Assay Method Information | |
| | In Vitro 17bHSD4 Enzyme Assay |
| Description: | 10 concentration of compounds (0.2 μl) in DMSO was added to GREINER FLUOTRAC 200 384 well plate (781076) using ECHO dispensing (BECKMAN COULTER). 80 nl of 10 mM Estradiol (SIGMA, E8875) was added using Echo dispensing. The enzyme reaction was initiated by addition, using MULTIDROP COMBI dispensing (THERMO FISHER), of 40 μl of a mix containing recombinant 17bHSD4 (M1-N311) and NAD. Final assay conditions were 40 nM of 17bHSD4, 0.125 mM of NAD, 15 μM Estradiol and various concentrations of compound in buffer (5 mM EDTA (TEKNOVA E0306), 0.01% DDM (AFFYMETRIX D310) in 50 mM Tris-Cl, pH 7.4). After each addition plates were centrifuged for 1 min at 150×g (EPPENDORF, 5810R, A-4-81). NADH formation was measured by fluorescence intensity (FI) (Ex360/Em460) at time zero (to) and at 1.5 h (t1) in a PHERASTAR FSX (BMG LABTECH). FI for each sample was calculated as FI at t1 minus FI at t0. |
| Affinity data for this assay | |
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