The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device.
Various monosaccharides, oligosaccharides and small polysaccharides were labelled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and the resulting fluorescent derivatives were separated by high-resolution PAGE. The electrophoretic mobilities of the labelled saccharides are related largely to the compounds' Mr values, but they are also influenced by the individual chemical structures of the saccharides. Various positional isomers and some epimers, for instance galactose and glucose, were resolved. Oligosaccharide and small polysaccharide derivatives, prepared from an enzymic digest of starch, each differing in size by a single hexose residue and with a range of degrees of polymerization from 2 to 26, were all resolved in a single gel. The method was relatively rapid and simple to perform. It enabled multiple samples to be analysed in parallel with high sensitivity. The fluorescent-labelling procedure was virtually quantitative. As little as 1 pmol of ANTS-labelled saccharide was detected photographically when the gels were illuminated by u.v. light. When the gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrate moieties of glycoconjugates and other naturally occurring oligosaccharides.
Jackson
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Astromed Ltd., Innovation Centre, Cambridge, U.K.
D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D008969:Molecular Sequence Data; D009281:Naphthalenes; D013696:Temperature
D002241:Carbohydrates; D005456:Fluorescent Dyes; D009281:Naphthalenes; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid