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.
Swartz, Darl R.
"Covalent Labeling of Proteins with Fluorescent Compounds for Imaging Applications,"
Scanning Microscopy: Vol. 1996
, Article 22.
Available at: https://digitalcommons.usu.edu/microscopy/vol1996/iss10/22