High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate.
An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.
D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006023:Glycoproteins / Q000097:blood*; D007834:Lasers; D013194:Staining and Labeling; D013861:Thiocyanates; D014466:Ultraviolet Rays
D005452:Fluoresceins; D006023:Glycoproteins; D013861:Thiocyanates; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate