| Assay Method Information | |
| | Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Vitamin D Receptor (VDR) Coactivator Assay |
| Description: | A VDR ligand binding domain tagged with GST (VDR-LBD(GST)), a fluorescein-TRAP220/DRIP-2-bound coactivator peptide (Fluorescein-peptide), LanthaScreen Tb-anti-GST (Goat) antibody (Tb-anti-GST), TR-FRET co-regulator buffer G, and DTT solution were used from LanthaScreen TR-FRET VDR Coactivator Assay Kit, which was purchased from Invitrogen. Each of the compounds synthesized as described above was dissolved in dimethyl sulfoxide (DMSO for molecular biology, Sigma Aldrich). The solution was diluted to a desired concentration with TR-FRET co-regulator buffer G containing 1 mass % of DMSO. The receptor-tracer-antibody complex solution was added to the compound solution in each well (20 μL) such that each well contained 1.0 nM of VDR-LBD(GST), 2.0 nM of Tb-anti-GST, and 100 nM of Fluorescein-peptide. The resulting mixture was incubated at room temperature for 2 hours. Each well was measured for TR-FRET using a microplate reader (Infinite F200 PRO, Tecan) equipped with an excitation filter at 340 nm (30 nm bandwidth), a terbium emission filter at 495 nm (10 nm bandwidth), and a tracer emission filter at 520 nm (25 nm bandwidth). On the basis of the resulting data, the 50% effective concentration (EC50) of each compound was calculated and evaluated using a graph plotting program (GraphPad Prism ver. 8.2.0) with the saturated activity of natural active vitamin D3 being normalized to 100%. |
| Affinity data for this assay | |
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