Fluorescence Anisotropy and Polarization in the Characterization of Biomolecular Association Processes and Their Application to Study SH2 Domain Binding Affinity

Abstract

Fluorescence anisotropy (or polarization) is a powerful technique to study biomolecular association processes, by following the rotational motions of one of the two partners in the interaction, labeled with a fluorophore. It can be used to determine dissociation constants in solution, down to nM values, and unlabeled ligands can be characterized, too, by using competition experiments. In this chapter, we introduce the basic principles of the technique, compare it with other experimental approaches, and discuss the experimental details with specific examples regarding SH2 domain/phosphopeptide association processes. The experimental protocols to be used in binding experiments and displacement studies are described, as well as the caveats to be considered in performing accurate measurements.