| Assay Method Information | |
| | PXR BINDING ASSAY |
| Description: | The time-resolved fluorescence resonance transfer (TR-FRET) hPXR competitive binding assay was performed according to the manufacturer's instructions (Invitrogen) with minor modifications. Briefly, the binding assays were performed in 384-well low volume (20 μl per well) solid black plates with 5 nM GST-hPXR ligand-binding domain, 40 nM fluorescent-labeled hPXR ligand (Fluormore PXR Green, also referred to as a tracer ), 5 nM terbium-labeled anti-GST antibody, and test compound at a variety of concentrations. DMSO (0.4%) was used as the negative control (0% inhibition) and a potent hPXR agonist, SR-12813 at 10 μM (with 0.4% DMSO) was used as the positive control (100% inhibition). The activities of individual chemicals tested at various concentrations (1-to-2 series dilutions for 16 concentration levels from 40 μM with 0.4% final DMSO concentration) were normalized to positive and negative controls to generate % Inhibition (PMID: 18784074).In the reaction mixture, GST-hPXR forms a complex with the terbium-labeled anti-GST antibody and the tracer. Excitation of terbium (the donor) using a 340-nm excitation filter results in energy transfer to the fluorophore of the tracer. This energy transfer is detected by an increase in the fluorescence emission of the tracer at 520 nm and a decrease in the fluorescence emission of terbium at 495 nm. The FRET ratio was calculated by dividing the emission signal at 520 nm by the emission signal at 495 nm. A competitor compound such as SR-12813 replaces the tracer from the complex and decreases the FRET ratio accordingly. The reactions were incubated at 25° C. for 30 min before measuring the fluorescent emission of each well at 495 and 520 nm using a 340-nm excitation filter, 100-μs delay time, and 200-μs integration time, with a PHERAStar plate reader (BMG Labtech, Durham, N.C.). The curve-fitting software GraphPad Prism 4.0 (Graphpad Software, La Jolla, Calif.) was used to generate the dose response curves and determine the IC50 values for individually tested compounds if applicable. SR-12813 has an IC50 of 65 nM in this assay. |
| Affinity data for this assay | |
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