Bradford Coomassie brilliant blue G-250 protein-binding dye exists in three forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromolecular form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compounds are explained in terms of their effects upon the equilibria between the three dye forms.