| Assay Method Information | |
| | HSD17B13 Inhibition Assay |
| Description: | HSD17B13 uses the oxidized form of nicotinamide adenine dinucleotide (NAD+) as a cofactor during metabolism of beta-estradiol (substrate), converting NAD+ to the reduced form (NADH) and beta-estradiol to its product (estrone). Estrone production is quantified by LCMS, and used as a measure of HSD17B13 enzyme activity.HEK-cells stably expressing wild type human HSD17B13 were plated at 10,000 cells/well in 50 μL growth media (DMEM containing 10% heat inactivated FBS, 400 μg/ml geneticin, 1× L-Glutamine, and 1× Non-essential amino acids, Invitrogen 11965-092, 16140-071, 10131-027, 25030-081, 11140-050), into poly-D-lysine-coated 384-well plates (Corning Biocoat, 354663), and incubated overnight (with lid) at 37° C. (95% O2: 5% CO2). Following overnight incubation, intermediate compound plates (Greiner, 781280) containing 160 nL of 375×FAC test compound which had been serial diluted 1 in 3.162 in 100% DMSO for an 11 point concentration response curve, with duplicate points at each concentration, were diluted 1 in 187.5 with 30 μL warmed assay media (DMEM, 1× L-Glutamine, and 1× Non-essential amino acids) to give 2×FAC compound (80 μM top concentration) in 0.53% DMSO. Growth media was then removed from the cell plates and replaced with 10 μL of 2×FAC test compound, and incubated for 1 hour (with lid) at 37° C. (95% O2: 5% CO2), before the addition of 25 μM FAC beta-estradiol in assay media/0.2% DMSO. The reaction was incubated for 2 hours (with lid) at 37° C. (95% O2: 5% CO2), after which 10 μL of reaction was transferred from assay plate to a new 384-well deep-well plate (Matrix 4325) and diluted 1 in 10 with 90 μL of stop reagent (50% methanol in water containing internal standard 17b-estradiol-2,3,4-13C3). Amount of product (estrone) was then quantified by LCMS. Data expressed as product area ratio (PAR) were then normalized to control wells using Activity Base (IDBS). |
| Affinity data for this assay | |
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