Surface plasmon resonance (SPR) is a sensitive analytical technique which allows the measurement of biomolecular interactions in real time. Protein-protein, small molecule-protein, nucleic acid-protein, carbohydrate-protein, peptide-protein and small molecule-nucleic acid interactions can all be characterised by SPR. One of the binding partners is captured or tethered in a 3D network, typically composed of carboxymethyl-dextran attached to a sensor chip surface, while the other binding partner is flowed across the surface. Binding of molecules close to the chip surface cause changes in the refractive index, which are detected by the instrument in real time. This allows for the determination of the kinetics and affinity of the interaction, by fitting the data to either kinetic models or equilibrium binding models.
Many methods are available to tether one of the binding partners to the sensor chip, including direct amine coupling, streptavidin capture of biotinylated biomolecules, nickel affinity capture of His tagged proteins and protein G affinity capture of antibodies. Optimization is required to determine the best immobilization method, buffer conditions, and assay method, in order to achieve the highest quality data possible.
Here we show examples of optimization necessary for high-quality SPR data and how SPR can be used to characterize protein-protein interactions, small molecule-protein interactions and antigen-antibody interactions.