Competitive SPR chaser assay to study biomolecular interactions with very slow binding dissociation rate constant

Binding kinetics of drug and its target protein is crucial for the efficacy and safety of the drug. Using surface plasmon resonance (SPR) technology, we performed a competitive SPR chaser assay, a method to study biomolecular interactions with very slow dissociation rate constants (k_d < 1E-4 s^-1). This report described the principle and the experimental setup of the chaser assay, which involves using a competitive probe (chaser) to detect changes in target occupancy by a test molecule over time. We demonstrated the applicability of the chaser assay for both small and large molecules and compared the results with conventional SPR kinetic analysis and other methods. We suggest that the chaser assay is a useful and robust technique to characterize very tight biomolecular interactions, and that it can also be used to study cooperativity in ternary complex formation.