Conjugation of fluorophores to tubulin

The use of fluorescently labeled proteins as probes of the structure and function of living cells began with the study of the components of the cytoskeleton(1-4) and has become a powerful approach for studying the distribution and dynamics of these components(5-7). Primary components of the cytoskeleton such as tubulin, actin and myosin are abundant and can be purified relatively easily in hundreds of milligram quantities from animal tissues(8-11). Fluorophores containing amine or thiol reactive groups are used to selectively modify Lysine or cysteine residues, respectively. It is essential that derivatization of the protein not result in toss of function. Methods for selecting active protein and removing unconjugated fluorophore are usually used after derivatization. The choice of fluorophore at any given wavelength of fluorescence emission should optimize fluorescence intensity and photostability. Tubulin is Labeled in polymeric form to protect residues that are important for assembly and can be successfully conjugated with fluorescent derivatives of amine-reactive succinimidyl esters. Functional tubulin is selected by cycles of polymerization and depolymerization. By modifying the ratio of reactants, the Cy3-labeling protocol can also be used to derivatize tubulin with Cy3.5, Cy5, tetramethylrhodamine, X-rhodamine, Oregon Green 488, and Alexa 488, 568 and 594 succinimidyl esters. Although fluorescent fusion proteins have become widely used in imaging studies, fluorophore-labeled proteins remain the most versatile and direct toots for studying the dynamics and function of the cytoskeleton. This protocol describes the fluorescent conjugation of tubulin isolated from brain tissue.