Detection of Target Proteins by Fluorescence Anisotropy

Understanding molecular interactions is critical to understanding most biological mechanisms of cells and organisms. In the case of small molecule–protein interactions, many molecules have significant biological activity through interactions with unknown target proteins and by unknown modes of action. Identifying these target proteins is of significant importance and ongoing work in our laboratories is developing a technique termed Dynamic Isoelectric Anisotropy Binding Ligand Assay (DIABLA) to meet this need. Work presented in this manuscript aims to characterize the fundamental parameters affecting the use of fluorescence anisotropy to detect target proteins for a given ligand. Emphasis is placed on evaluating the use of fluorescence anisotropy as a detection mechanism, including optimization factors that affect the protein detection limit. Effects of ligand concentration, pH, and nonspecific binding are also examined.