| Assay Method Information | |
| | FP Competitive Inhibition Assay |
| Description: | Experiments were performed in 96-well Microfluor 2 black plates on a Synergy 2 plate reader (Biotek). The polarization was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. The FP experiments were performed in an assay buffer of 25 mM Hepes (pH 7.4), 100 mM NaCl, 0.01% Triton X-100, and 100 μg/ml γ-globulin. The final reaction volume was set to 100 μL. For the β-catenin/Tcf assay, 10 nM human β-catenin (residues 138-781) was incubated with 2.5 nM C-terminally fluorescein-labeled human Tcf4 (residues 7-51) for 30 min at 4° C., and then different concentrations of the compound in assay buffer were added. The negative control (equivalent to 0% inhibition) refers to 2.5 nM Tcf4 fluorescence tracer and 10 nM β-catenin in assay buffer without the tested compound. The positive control (equivalent to 100% inhibition) refers to only 2.5 nM Tcf4 fluorescence tracer in assay buffer. For the β-catenin/cadherin assay, 150 nM human i-catenin (residues 138-781) was incubated with 5 nM C-terminally fluorescent-labeled human E-cadherin (residues 819-873) in assay buffer for 30 min at 4° C. The negative control refers to 5 nM E-cadherin fluorescence tracer and 150 nM β-catenin in assay buffer with no inhibitor presenting. The positive control refers to 5 nM E-cadherin fluorescence tracer in assay buffer. For the β-catenin/APC-R3 assay, 2000 nM human β-catenin (residues 138-781) was incubated with 5 nM of C-terminally fluorescent-labeled human APC-R3 (residues 1477-1519) in assay buffer for 30 min at 4° C. The negative control refers to 5 nM APC-R3 fluorescence tracer and 2,000 nM β-catenin in assay buffer without the tested compound. The positive control refers to 5 nM APC-R3 fluorescence tracer in assay buffer. Each assay plate was covered black and gently mixed on an orbital shaker at 4° C. for 2.5 h to reach equilibrium before the polarization values were read. The background of the tested inhibitors was corrected by subtracting the raw intensity values of the sample background well (all components except probe) from the raw intensity values of the corresponding test wells (all components). |
| Affinity data for this assay | |
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