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. 1983

R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci

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.

UI MeSH Term Description Entries
D007834 Lasers An optical source that emits photons in a coherent beam. Light Amplification by Stimulated Emission of Radiation (LASER) is brought about using devices that transform light of varying frequencies into a single intense, nearly nondivergent beam of monochromatic radiation. Lasers operate in the infrared, visible, ultraviolet, or X-ray regions of the spectrum. Masers,Continuous Wave Lasers,Pulsed Lasers,Q-Switched Lasers,Continuous Wave Laser,Laser,Laser, Continuous Wave,Laser, Pulsed,Laser, Q-Switched,Lasers, Continuous Wave,Lasers, Pulsed,Lasers, Q-Switched,Maser,Pulsed Laser,Q Switched Lasers,Q-Switched Laser
D003208 Concanavalin A A MANNOSE/GLUCOSE binding lectin isolated from the jack bean (Canavalia ensiformis). It is a potent mitogen used to stimulate cell proliferation in lymphocytes, primarily T-lymphocyte, cultures.
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D005452 Fluoresceins A family of spiro(isobenzofuran-1(3H),9'-(9H)xanthen)-3-one derivatives. These are used as dyes, as indicators for various metals, and as fluorescent labels in immunoassays. Tetraiodofluorescein
D006023 Glycoproteins Conjugated protein-carbohydrate compounds including MUCINS; mucoid, and AMYLOID glycoproteins. C-Glycosylated Proteins,Glycosylated Protein,Glycosylated Proteins,N-Glycosylated Proteins,O-Glycosylated Proteins,Glycoprotein,Neoglycoproteins,Protein, Glycosylated,Proteins, C-Glycosylated,Proteins, Glycosylated,Proteins, N-Glycosylated,Proteins, O-Glycosylated
D013194 Staining and Labeling The marking of biological material with a dye or other reagent for the purpose of identifying and quantitating components of tissues, cells or their extracts. Histological Labeling,Staining,Histological Labelings,Labeling and Staining,Labeling, Histological,Labelings, Histological,Stainings
D013861 Thiocyanates Organic derivatives of thiocyanic acid which contain the general formula R-SCN. Rhodanate,Rhodanates
D014466 Ultraviolet Rays That portion of the electromagnetic spectrum immediately below the visible range and extending into the x-ray frequencies. The longer wavelengths (near-UV or biotic or vital rays) are necessary for the endogenous synthesis of vitamin D and are also called antirachitic rays; the shorter, ionizing wavelengths (far-UV or abiotic or extravital rays) are viricidal, bactericidal, mutagenic, and carcinogenic and are used as disinfectants. Actinic Rays,Black Light, Ultraviolet,UV Light,UV Radiation,Ultra-Violet Rays,Ultraviolet Light,Ultraviolet Radiation,Actinic Ray,Light, UV,Light, Ultraviolet,Radiation, UV,Radiation, Ultraviolet,Ray, Actinic,Ray, Ultra-Violet,Ray, Ultraviolet,Ultra Violet Rays,Ultra-Violet Ray,Ultraviolet Black Light,Ultraviolet Black Lights,Ultraviolet Radiations,Ultraviolet Ray
D016650 Fluorescein-5-isothiocyanate Fluorescent probe capable of being conjugated to tissue and proteins. It is used as a label in fluorescent antibody staining procedures as well as protein- and amino acid-binding techniques. FITC,5-Isothiocyanatofluorescein,Fluorescein (5 or 6)-Isothiocyanate,Fluorescein-5-isothiocyanate Hydrochloride,5 Isothiocyanatofluorescein,Fluorescein 5 isothiocyanate,Fluorescein 5 isothiocyanate Hydrochloride,Hydrochloride, Fluorescein-5-isothiocyanate

Related Publications

R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
October 1979, Hoppe-Seyler's Zeitschrift fur physiologische Chemie,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
March 1974, Analytical biochemistry,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
September 1978, Analytical biochemistry,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
May 1976, Analytical biochemistry,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
January 1980, Acta biologica et medica Germanica,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
April 1990, BioTechniques,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
January 1988, Nauchnye doklady vysshei shkoly. Biologicheskie nauki,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
November 1975, Analytical biochemistry,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
May 1991, Analytical biochemistry,
R Cotrufo, and M R Monsurrò, and G Delfino, and G Geraci
November 1985, Analytical biochemistry,
Copied contents to your clipboard!