| Assay Method Information | |
| | Human IL-17A x IL-17RA TR-FRET assay |
| Description: | Dilutions of test compounds in DMSO are plated onto white low-volume 384-well plates (Greiner, #784075). Specifically, solutions of graded concentrations (5 mM to 150 nM; sixteen 2-fold dilution steps) are dispensed at 0.2 µL/well to yield final assay concentrations in the range of 100 µM to 3 nM. Each compound concentration is tested in duplicate.All components of the assay are diluted in PBS containing 0.05% Tween20 (Sigma, #P1379, 5% stock in water), 1 mM EDTA (Invitrogen, #15575-038; 0.5M stock, pH8) and 0.05% bovine serum albumin (Roche; 5% stock in water). The final total assay volume is 10 µL. Reagents are added using a Multidrop Combi device (ThermoFisher, #5840320); between reagents, the instrument is rinsed with buffer:(i) Biotinylated human IL-17A (PN106276; stock solution: 55.87 µM) is added at 5 µL/well from a 10 nM intermediate dilution to yield a final assay concentration of 5 nM. Each assay plate contains low controls where buffer is added in place of IL-17A.(ii) After 30 minutes of incubation at RT, 2.5 µL/well of AF647-labeled human IL-17RA (PN110235; stock: 2.91 µM) is added from a 120 nM intermediate solution, to yield a final assay concentration of 30 nM.(iii) Following 30 minutes of incubation at RT, 2.5 µL/well of Eu-Streptavidin (PerkinElmer, #AD0062; stock: 500 µg/mL=8.3 µM) is added from an intermediate dilution of 4 nM to yield a final concentration of 1 nM.After 30 to 60 minutes of incubation, FRET is measured in an EnVision 2101 Multilabel-Reader (Perkin Elmer) with excitation at 320 nm, and emission at 615 and 665 nm (program TR-FRET Screening , instrument: NIBR-00013163).Each plate contains test compounds in columns 1-22 and controls in columns 23 and 24. The neutral control (NC) contains all the assay components, but DMSO instead of test compound. The active control (AC) mimics complete inhibition of the IL-17 ligand interaction, which is achieved by replacing the biotinylated cytokine with buffer. The fluorescence values for emission at 615 nm and 665 nm are loaded to the evaluation software Helios. |
| Affinity data for this assay | |
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