| Assay Method Information | |
| | In Vitro Activity Assay |
| Description: | Table 1: Experimental materials: target protein RORγ with the final concentration of 200 nM; experimental buffer (10×) MOPS (500 mM) PH 7.4, CHAPS (0.5 mM), NaF (500 mM), andBSA (1 mg/ml); donor microbeads in the kit with the final concentration of 5 μg/mL, and acceptor microbeads with the final concentration of 5 μg/mL; co-agonist of RORγ, short peptide bSRC1-4(Biotin-QKPTSGPQTPQAQQKSLLQQLLTE), with the final concentration of 50 nM. 150 μL of reaction system: RORγ 15 μL, experimental buffer 15 μL, deionized water 60 μL, small molecule compound 15 μL, donor microbeads 15 μL, and acceptor microbeads 15 μL; positive inhibitors T0901317 and UA.Experimental method: The protein, co-agonist (b-SRC1-4), 10× AlphaScreen buffer, and ultra-pure water were prepared into a mixed solution with the final volumes of 15 μL, 15 μL, 15 μL, and 60 μL, respectively (the final concentration ratio of protein to co-agonist is 200:50 nM). 105 μL of the mixed solution was added to each sample to be tested in a 96-well transparent plate. If the single-point inhibition rate of the compound was tested, the compound was diluted to the final concentration of 50 μM, and 15 μL of the diluted compound was added to each sample. If the IC50 value of the compound was tested, the compound was doubling diluted (to 200 to 0.075 μM), and 15 μL of the diluted compound was added to each sample (generally, in order to save manpower and material resources, a batch of new compounds were subjected to single-point preliminary screening, and then compounds with an inhibition rate of about 50% were tested for IC50 curves). The donor microbeads and acceptor microbeads in the final concentration of 5000 g/mL should be prepared to 5 g/mL, and in the green light environment, 30 μL of the mixed solution of the two microbeads was added to each well. The mixture was centrifuged at room temperature at 1000 rpm for 1 minute to make the system fully mixed. After being wrapped in tin foil, the mixture was incubated in the dark for 1.5 hours. After that, the mixture was transferred to a 384-well white opaque plate and put into an EnSpire Alpha 2390 multifunctional microplate reader to detect the inhibitory activity of the compound. |
| Affinity data for this assay | |
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