The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels.
Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.
D055598:Chemical Phenomena; D002621:Chemistry; D003208:Concanavalin A / Q000138:chemical synthesis*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005452:Fluoresceins / Q000138:chemical synthesis*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006023:Glycoproteins / Q000032:analysis*; D013053:Spectrophotometry
D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D003208:Concanavalin A; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein