Protein-Protein Interactions

Zaneta Nikolovska-Coleska, Nikolovska-Coleska, Zaneta

Protein-protein interactions regulate many important cellular processes, including carbohydrate and lipid metabolism, cell cycle and cell death regulation, protein and nucleic acid metabolism, signal transduction, and cellular architecture. A complete understanding of cellular function depends on full characterization of the complex network of cellular protein-protein interactions, including measurements of their kinetic and binding properties. Surface plasmon resonance (SPR) is one of the commonly used technologies for detailed and quantitative studies of protein-protein interactions and determination of their equilibrium and kinetic parameters. SPR provides excellent instrumentation for a label-free, real-time investigation of protein-protein interactions. This chapter details the experimental design and proper use of the instrumentation for a kinetic experiment. It will provide readers with basic theory, assay setup, and the proper way of reporting this type of results with practical tips useful for SPR-based studies. A generic protocol for immobilizing ligands using amino coupling chemistry, also useful if an antibody affinity capture approach is used, performing kinetic studies, and collecting and analyzing data is described.