Updates to Guidance on Using Biacore for Protein-Protein Interactions Analysis

The use of optical biosensors for studying macromolecular interactions is now considered essential technology for understanding the biophysical properties of potential therapeutic and diagnostic reagents. Indeed, according to Cytiva (2025, Key-SPR-applications. https://www.cytivalifesciences.com/en/us/solutions/protein-research/Interaction-analysis-with-Biacore-surface-plasmon-resonance-SPR/Key-SPR-applications ), over 50 000 Biacore papers have been published to date, the sheer volume and variety of which present a daunting task for the burgeoning Biacore user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. This document is written to guide the Biacore user through basic theory, system maintenance, and assay setup, while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterization of single-chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Although we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilized surface prior to kinetic analysis, these methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.