| Assay Method Information | |
| | 3β-HSD1 Inhibition Assay |
| Description: | DHEA and NAD+ were enzymatically converted to androstenedione and NADH, respectively. Final NADH concentrations were spectroscopically quantified using a commercially available Amplite™ Colorimetric NADH Assay Kit which utilizes an NADH probe with an absorbance wavelength of 460 nm. Enzymatic assays with a final volume of 25 μL were ran in a 384-well, Greiner Bio-One UV-STAR® assay plate in 50 mM TRIS pH 8.0 assay buffer. Compound IC50 values were experimentally determined in duplicate using a 10-point concentration response curve (CRC) with a top concentration of 50 μM and a bottom concentration of 25 pM. Compound stocks at 10 mM in DMSO were serial diluted 5-fold in DMSO to generate an ECHO® compatible source plate. 125 nL of DMSO, Trilostane, or serial diluted compounds were then acoustically transferred into an empty UV-STAR® assay plate to give a final assay concentration of 0.5% v/v DMSO. The stamp volume and dilution scheme can be adjusted to accommodate compound potency. The assay can tolerate up to a 5% v/v DMSO final assay concentration. 15 μL of 1 μg purified 3β-HSD1 and 50 μM DHEA (final assay volume concentration) was dispensed per well into the stamped plate, centrifuged for 1 minute at 1000 RPM, covered, and allowed to incubate for 15 minutes at room temp. Enzymatic reactions were initiated upon the addition of 10 μL of NAD+ diluted in assay buffer to give a final assay volume concentration of 2 mM. The assay plate was centrifuged for 1 minute at 1000 RPM, covered, and incubated at room temperature for 1 hour. 25 μL Amplite™ Colorimetric NADH Assay Kit detection solution (prepared as recommended) was added to all reaction wells, the plate centrifuged for 1 minute at 1000 RPM, covered, and incubated for 15 minutes. Final absorbance values (λ=460 nm) are spectroscopically determined on BioTek® Cytation5 or Synergy4 plate readers. The background signal was determined by averaging 32 fully inhibited reactions (Trilostane treated) and subtracted across the plate. From the background subtracted values, the non-inhibited enzyme signal (DMSO treated) was averaged from 32 control reactions. |
| Affinity data for this assay | |
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