| Assay Method Information | |
| | FXR Biochemical Assay |
| Description: | Purpose of the Assay:Activation of FXR binding reaction by the compound was detected by the amplified luminescent proximity homogeneous assay (alphascreen).Materials of the Assay:1. Protein: Glutathione-S-transferase-labeled FXR human protein (Invitrogen)2. Coactivator: Biotin-labeled steroid receptor coactivator (Anaspec)3. Detection reagent: Detection kit for the amplified luminescent proximity homogeneous assay (alphascreen) (PerkinElmer)Method of the Assay:1. Dilution of the compound: The test compound was prepared as a 40 μM DMSO solution, and then diluted 3-fold to 10 concentration points. The reference compound was prepared as a 400 μM DMSO solution, and then diluted 1.5-fold to 10 concentration points. The diluted DMSO solution was added to the microwells of a 384-well plate at a volume of 150 nl per well.2. The Glutathione-S-transferase-labeled FXR human protein and the biotin-labeled steroid receptor coactivator were prepared into mixed solutions with concentrations of 0.4 nM and 30 nM, respectively. The mixed solutions were added to the microwells of the 384-well plate at a volume of 15 μL per well, and incubated for 1 hour at room temperature.3. The mixture solution of receptor beads in the detection kit for the amplified luminescent proximity homogeneous assay (alphascreen) was diluted 125-fold, and added to the microwells of the 384-well plate at a volume of 7.5 μL per well. The operation during the assay process was protected from light. The incubation was perfomed at room temperature for 1 hour.4. The mixture solution of donor beads in the detection kit for the amplified luminescent proximity homogeneous assay (alphascreen) was diluted 125-fold, and added to the microwells of the 384-well plate at a volume of 7.5 μL per well. The operation during the assay process was protected from light. The incubation was perfomed at room temperature for 1 hour.5. EC50 assay: Envision was used with excitation at 680 nm, and the absorption signal was read at 520-620 nm.6. Analysis of the data: The data were analyzed with Prism 5.0. |
| Affinity data for this assay | |
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