Assay Method Information

Assay Name:  Fluorescence Resonance Energy Transfer (FRET) Assay
Description:  The inhibitory activity of compounds on RORγ receptor was determined by fluorescence resonance energy transfer (FRET) experiments. The inhibitory activity was expressed by half-inhibitory concentration (IC50).Experiment Method1. Preparation of RORγ Buffer Solution10 mL of DTT and 100 mL buffer solution were gently mixed together and ready to use.2. Preparation of Compound SolutionThe concentration of compound solution started from 7.5 mM to 0.25 mM, which was diluted by every 3 folds from 7.5 nM with 10 concentrations totally.3. Preparation of Protein Mixturea. 40 nM B-RORγ LBD solution and 20 nM SA-APC solution were gently mixed together. The reaction mixture was incubated for 15 minutes at room temperature. Then 400 nM Biotin was added to the described above mixture. After gently mixed together, the resulting mixture was incubated for 10 minutes at room temperature.b. 40 nM Bioin-SRC1 solution and 10 nM SA-eu solution were gently mixed together, and the reaction mixture was incubated for 15 minutes at room temperature. Then 200 nM Biotin was added to the described above mixture. After gently mixed together, the resulting mixture was incubated for 10 minutes at room temperature.c. The described above two pre-mixes were gently mixed together with a ratio of 1:1, and the resulting mixture was incubated for 5 minutes at room temperature.d. A mixture of 0.1 μM alternative agonist N-(2-chloro-6-fluorophenyl)-N-((20-methoxy-[1,10-biphenylyl]-4-substituted)methyl)benzenesulfonamide, 25 μL B-RORγ LBD/SA-APC and Bioin-SRC1/SA-eu mixture solution with test compound were added to one of the well of a 384-well plate, then they were centrifuged for 1 minute at 1000 rpm, and incubated at room temperature for 1 hour.
Affinity data for this assay
 

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