Laser excitation of fluorescent-labeled polypeptides in polyacrylamide gels.
A laser beam at 488 nm, converted into a fan of light by a surface-coated mirror oscillated in response to a triangular wave, was inserted into the base of a polyacrylamide gel. The laser light was trapped by internal reflection and gave uniform illumination throughout the entire gel slab. Photography with color film detected 50 fmol of fluorescein covalently coupled to ovalbumin, gave 80-fold greater sensitivity than transillumination in detection of fluorescein-labeled polypeptides, and was about 25-fold more sensitive than protein staining with silver. Laser illumination visualized end-labeled beta-galactosidase, afforded quality control of such preparations, and demonstrated that the end-labeled derivative contained about 25-fold less fluorescein than uniformly labeled beta-galactosidase. The latter result was confirmed by dot-blot analysis using a polyclonal antibody specific for fluorescein. The application of end-labeling to the location of features of protein primary structure is discussed.
Duffy
V E
VE
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Stockwell
P A
PA
Warrington
D M
DM
Monk
B C
BC
D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007834:Lasers; D010455:Peptides; D013861:Thiocyanates; D001616:beta-Galactosidase
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010455:Peptides; D013861:Thiocyanates; D001616:beta-Galactosidase; D016650:Fluorescein-5-isothiocyanate