| Assay Method Information | |
| | Fluorescence Polarization (FP) Assay |
| Description: | The FP experiments were performed in 96-well Microfluor 2 black plates (Waltham, MA), and the sample signals were read by a Synergy 2 plate reader (Biotek, Winooski, VT). The polarization was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. All of the FP experiments were performed in an assay buffer of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 100 pug/mL of bovine γ-globulin, and 0.010% Triton X-100. The final reaction volume was set to 100 μL. In the FP saturation binding experiments, the concentration of human BCL9 fluorescent tracer was fixed at 5 nM. The concentrations of β-catenin were ranged from 0 to 10 μM in the assay buffer giving a final volume of 100 μL. After the addition, each assay plate was covered black and gently mixed on an orbital shaker for 3 h before the polarization signals were recorded. The data were analyzed by nonlinear least-square analyses using GraphPad Prism 5.0 to derive the apparent Kd value. Each experiment was repeated three times, and the results were expressed as mean±standard deviation. |
| Affinity data for this assay | |
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