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.
Swartz
D R
DR
Department of Anatomy, Indiana University Medical Center, Indianapolis 46202. dswartz@indyvax.iupui.edu
D000185:Actinin / Q000032:analysis / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016591:Histocytological Preparation Techniques; D008856:Microscopy, Fluorescence / Q000379:methods*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000648:ultrastructure; D009210:Myofibrils / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000185:Actinin; C015296:5-iodoacetamidofluorescein; C037041:fluorescein 5-maleimide