Covalent labeling of proteins with fluorescent compounds for imaging applications

The labeling of proteins with fluorescent compounds for microscopy has allowed a greater understanding of biological processes. The preparation of fluorescent proteins is the first step in development of their use in microscopy. Methods are described to label and characterize a protein as an example of the general approach for other proteins. Skeletal muscle alpha-actinin was labeled with either fluorescein-5-maleimide or 5-iodoaceamidofluorescein and the reaction characterized. The maleimide reaction was much more rapid and efficient than the iodoacetamide reaction giving a coupling efficiency of 65% under the given ration conditions. The fluorescein-5-maleimide alpha-actinin was functionally characterized and there was essentially no influence on the fluorescein label on the F-actin binding properties of alpha-actinin. The fluorescein alpha-actinin was also shown to specifically bind to the Z-line of isolated myofibrils. A general outline and discussion are presented on how to label and characterize proteins for use in microscopy.