FP assays were carried out essentially as described previously.39 (link) Briefly, an appropriate 5-carboxyfluorescein-labeled PBD-binding peptide for Plk1, Plk2, or Plk3 was incubated at a concentrations of the bacterially expressed and purified PBD of Plk1, Plk2, or Plk3, respectively, in a binding buffer containing 10 mM Tris (pH 8.0), 1 mM EDTA, 50 mM NaCl, and 0.01% Nonidet P-40. FP was analyzed 10 min after mixing of all components in a 384-well format using a Molecular Devices SpectraMax Paradigm Multi-Mode Micro-plate Detection Platform. All experiments were performed in triplicate. Obtained data were plotted using GraphPad Prism software version 6.
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Fluorescence Polarization Assay for PLK Binding
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Corresponding Organization : Center for Cancer Research
Other organizations : Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Dongguk University, Korea University
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Variable analysis
independent variables
- Concentrations of the bacterially expressed and purified PBD of Plk1, Plk2, or Plk3
- Fluorescence polarization (FP)
- Binding buffer containing 10 mM Tris (pH 8.0), 1 mM EDTA, 50 mM NaCl, and 0.01% Nonidet P-40
- Appropriate 5-carboxyfluorescein-labeled PBD-binding peptide for Plk1, Plk2, or Plk3
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