| Assay Method Information | |
| | Nrf2-Keap1 FP Assay #2 |
| Description: | 10-point semi-log dilution curves in DMSO are prepared in 96-well polypropylene plates to 100× final assay concentration. Compound curves are then diluted 10-fold in assay buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 2 mM CHAPS, and 0.005% BSA). To a Costar 384-well black polystyrene assay plate (Corning, #3573), 5 μL of the 10× compound dose response curves are added, with columns 1, 12, 13, and 24 receiving only DMSO. The top concentration of compound is located in columns 2 and 14. Keap1 (321-609) is diluted to 17.5 nM (2.5×) in assay buffer and 20 μL is added to all wells except columns 12 and 24 (rows A-H), which get 20 μL of assay buffer instead. Immediately, 25 μL of 4 nM (2×) Tamara labeled peptide (AFFAQLQLDEETGEFL, 21st Century Biochemicals) in assay buffer is added to all wells except columns 12 and 24 (rows I-P), which get 25 μL of assay buffer instead. The assay plate is sealed with a foil film, shaken at 600 rpm for 1 h at room temperature and then read on a Pherastar (BMG), equipped with a TAMRA FP optic module (540/20 nm excitation and 590/20 nm emission filters). Fluorescence measurements, represented as mP, are used in the transformation of the data. The final assay concentrations of Keap1 (321-609) and Tamra labeled peptide are 7 nM and 2 nM, respectively. For each plate, the Pherastar's gain is adjusted after setting the min to 35 mP. The average background reading is automatically subtracted from each sample. Compound activity is calculated based on percent inhibition, normalized against controls in the assay (Control 1 contains the Tamra peptide and Keap1 (321-609) together (0% response) and Control 2 contains the Tamra peptide alone (100% response)). Control 3, containing only Keap1 (321-609) in columns 12 and 24 (rows I-P) are used for background. Data analysis is handled in Prism by first transforming the % inhibition values using x=log x and then using the nonlinear regression equation sigmoidal dose-response curve (variable slope) to determine the pIC50 values. The % inhibition=100*(1−((compound response−average min)/(average max−average min))). |
| Affinity data for this assay | |
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