| Assay Method Information | |
| | In Vitro 17bHSD13 Enzyme Assay |
| Description: | Final assay conditions were 80 nM of 17bHSD13, 0.5 mM of NAD, 20 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 2.5 h the reaction were stopped by addition of 20 μl of 0.6% Formic acid (MERCK 5.33002) and samples were analyzed using LC-MS/MS.SCIEX LC-MS/MS system: Sample was injected with CTC analytical injector, SHIMATZU LC pumps LC20 and analyzed on the SCIEX API 5000 LCMSMS system with the following settings. Samples were chromatographed on a WATERS, SYMMETRY, C8, 3.5 μm, 2.1×50 mm) column at constant flow rate of 0.5 mL/min. The mobile phases consist of A (water with 0.2% formic acid) and B (acetonitrile with 0.2% formic acid). The LC gradient profile is as follows: 50% B during 0 to 0.5 min, a linear increase to 100% B during 0.5 to 1 min, hold at 100% B during 1 to 1.6 min then back to 50% B from 1.6 to 2 min. The run time was 2 min with retention times of approximately 0.8 and 1.07 min for Estradiol and Estrone, respectively. Detection was performed on a API 5000 LC/MS/MS system with a triple quadrupole mass spectrometer, a TURBO V ion source, in multiple reaction monitoring (MRM) mode at positive polarity with APCI probe. The MRM pairs were m/z 273.1 to m/z 107.0 and m/z 271.3 to 107.0. for Estradiol and Estrone, respectively. |
| Affinity data for this assay | |
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