Surface Plasmon Resonance: A Sensitive Tool to Study Protein–Protein Interactions

Abstract

Surface plasmon resonance (SPR) is one of the most commonly used techniques to study protein–protein interactions. The main advantage of SPR is the ability of measuring binding affinities and association/dissociation kinetics of complexes in real time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the “ligand,” on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the “analyte,” over the surface containing the ligand. The binding is monitored by following changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. During the last 15 years, SPR has been intensively used in the study of bacterial secretion systems due to its ability of detecting highly dynamic complexes, which are difficult to investigate by other techniques. This chapter will guide users in setting up SPR experiments in order to identify protein complexes and to assess their binding affinity and/or kinetics. It will include detailed protocols for (i) immobilization of proteins with the amine coupling capture method, (ii) analyte-binding analysis, (iii) affinity/kinetics measurements, and (iv) data analysis.