| Assay Method Information | |
| | Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay |
| Description: | TR-FRET assay was performed on a Wallac Victor 3V multi-label counter/plate reader (PerkinElmer, Shelton, CT). The plate used for the FP measurement was 384-well plates (Corning® 384 Well Low Flange White Flat Bottom Polystyrene NBS™ Microplate), loaded with 20 μL of assay solution per well. The buffer used in FP assays is TR-FRET dilution buffer from ThermoFisher (PV3574). 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 20 μL containing 5 nM of Keap1, 25 nM of FITC-Nrf2-9mer-NH2 and 0.5 nM of Terbium labeled anti-HIS antibody+inhibitor compound of different concentrations in DMSO or DMSO alone (1% final concentration) in assay buffer in triplicate. 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 shaked for 60 mM at room temperature and then centrifuged for 2 mM and TR-FRET was measured on a Victor 3V microplate reader. After excitation at 340 nm, well fluorescence was monitored at 495 nm and 520 nm. 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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