| Assay Method Information | |
| | Biochemical AlphaScreen assay |
| Description: | All experiments were performed in shite opaque 384-well plates from PerkinElmer (Waltham, MA) with an assay buffer of 25 mM HEPES (pH=7.4), 100 Mm nAcL, 0.1% BSA, and 0.01% Triton X-100. All sample signals were read on a excitation wavelength was set at 680 nm and emission at 570 nm. All of the final reaction volumes were set to 25 μL. In the cross-titration experiments of the wild-type β-catenin//wild-ty-e BCL9 interaction, N-terminally biotinlyated BCL9 (from 0 to 60 nM) and C-terminally His6-tagged μcatenin (2.5, 5, 10, 20, 40, and 80 nM) were titrated in 20 μg/mL) were added. The mixture was then covered black and incubated at 4° C. for 1 h before detection. All addition and incubation was made under subdued lighting conditions due to the photosensitivity of the beads. The data were analyzed by nonlinear least-square analyses using GraphPad Prism 5.0. Each experiment was repeated three times, and the results were expressed as mean ±standard deviation. The competitive binding experiments were performed to determine the apparent Kd values. The rule of the competitive binding experiments for associating the IC50 value with the Kd value are: (1) the expected Kd value should be 10 times higher than the concentration of either tested protein: (2) the concentrations of both tested proteins should be lower than the binding capacities of their respective beads; and (3) the concentration of the target protein (His6-tagged β-catenin) should be 10 times lower than that of the ligand protein (biotinylated BCL9). In the completive binding experiments to determine the Kd value for β-catenin/BCL9 interactions, 5 nM of N-terminally biotinylated BCL9, 0.5 nM of C-terminally His6-tagged β-catenin, and different concentrations of unlabeled BCL9 peptide (0-50 μM) were incubated at 4° C. in 20 μL assay buffer for 2 h. The donor and acceptor beads were added to a final concentration of 10 μg/mL in 25 μL assay buffer. The mixture was covered black and incubated for 1 h at 4° C. before detection. The IC50 values, which sere also the apparent Kd values from the AlphaScreen assay, were determined by nonlinear least-square analyses using GraphPad Prism 5.0. Each experiment was repeated three times, and the results were expressed as mean ±standard deviation |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |