Biolayer interferometry for measuring the kinetics of protein-protein interactions and nanobody binding

Protein-protein interactions underpin nearly all biological processes, and understanding the molecular mechanisms that govern these interactions is crucial for the progress of biomedical sciences. The emergence of artificial intelligence-driven computational tools can help reshape the methods of structural biology; however, model data often require empirical validation. The large scale of predictive modeling data will therefore benefit from optimized methodologies for the high-throughput biochemical characterization of protein-protein interactions. Biolayer interferometry is one of very few approaches that can determine the rate of biomolecular interactions, called kinetics, and, of the commonly available kinetic measurement techniques, it is the most suitable for high-throughput experimental designs. Here we provide step-by-step instructions on how to perform kinetics experiments using biolayer interferometry. We further describe the basis and execution of competition and epitope binning experiments, which are particularly useful for antibody and nanobody screening applications. The procedure requires 3 h to complete and is suitable for users with minimal experience with biochemical techniques.