| Assay Method Information | |
| | Fluorescence Polarization (FP) Assay |
| Description: | Fluorescence polarization assay was performed on a Wallac Victor 3V multi-label counter/plate reader (PerkinElmer, Shelton, CT) using 484 nm excitation and 535 nm emission filters for the fluorophore used in the binding experiment. The plate used for the FP measurement was Corning 3575 384-well plate, loaded with 40 μL of assay solution per well. The buffer used in FP assays is 10 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 50 mM EDTA, and 0.005% Tween-20. Deionized water from a Millipore water purification used to prepare all aqueous solutions in FP assay. The fluorescent probe used in this assay is 9-mer Nrf2 ETGE motif derived peptide, FITC-LDEETGEFL-NH2. In each well, the final volume is 40 μL that consisted of 10 μL of 400 nM Keap1 Kelch domain protein, and 20 μL of 20 nM FITC-9mer Nrf2 peptide amide, and 10 μL of an inhibitor compound of different concentrations. The experiments were done in triplicates, with initial concentration of the inhibitor typically 5 μM and 50 μM. Then, the plate was centrifuged for 2 mM to ensure thorough mixing and get rid of any air bubbles in the solution. The plate was covered and shacked for 30 mM at room temperature and then centrifuged for 2 mM prior to FP measurements. The determination of FP is by measuring the parallel and perpendicular fluorescence intensity (F∥ and F⊥) with respect to the linearly polarized excitation light. The measurement of IC50 of the inhibitors was determined from the plot of % inhibition against concentration of the inhibitor analyzed by Sigma Plot 12.3 software. |
| Affinity data for this assay | |
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