Binding Affinity and Kinetic Analysis of Nuclear Receptor/Co-Regulator Interactions Using Surface Pl

Knowledge of the kinetics of protein—protein interactions has become important in defining nuclear receptor function. Such knowledge allows characterization of interactions that occur with high affinity and/or selectivity. Surface plasmon resonance is a useful and sensitive tool for studying protein—protein interactions. This technique involves immobilization of a “ligand” to the surface of a sensor chip and subsequently passing over multiple concentrations of “analyte” to generate binding curves. Interaction between the receptor and target protein is monitored by the density at the sensor chip surface and allows calculation of the association and dissociation stages (and therefore affinity) of interactions to be assessed in real-time. Using software packages, these kinetic parameters can be quantified. Importantly, the levels of recombinant protein required are much less than that needed for other affinity techniques such as isothermal titration calorimetry and anisotropy fluorescence spectroscopy.