| Assay Method Information | |
| | Biochemical Assay |
| Description: | A cell free coactivator recruitment assay was developed for FXR, to measure the functional potency of novel FXR compounds. In this assay, novel FXR compounds were bound to the ligand binding domain (LBD) of recombinant human FXR. The ability of this liganded homodimeric complex to recruit the co-activator protein steroid receptor coactivator 1 (SRC-1) was measured using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). The LBD of human FXR (amino acids 244-476) was cloned into an expression vector for protein expression in SF9 insect cells. Purified FXR LBD was incubated in TR-FRET coregulator buffer G (Eurofins) with test compounds (0-10 micromolar) and a labeled nuclear receptor interaction domain of SRC-1 at 25° C. for 60 minutes (final DMSO vehicle concentration was 1.2%). Assays were analyzed using an Envision TR-FRET reader (PerkinElmer Life and Analytical Sciences). Compound activity at each concentration was defined as a percentage of the maximal activity of the natural FXR ligand agonist chenodeoxycholic acid (CDCA) generating a dose response curve. |
| Affinity data for this assay | |
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