The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis |
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Authors: | Peter Jackson |
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Institution: | (1) Division of Transfusion Medicine, University of Cambridge, Long Road, CB2 2PT Cambridge, UK |
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Abstract: | The glycans of glycoconjugates mediate numerous important biological processes. Their separation and structural determination
present considerable difficulties because of the small quantities that are available from biological sources and the inherent
difficulty of analyzing the wide variety of complex structures that exist. A method for the analysis of reducing saccharides
by PAGE that uses specific fluorophore labeling and is simple, rapid, sensitive, and readily available to biological researchers,
has been developed. The method is known acronimically either as PAGEFS (PAGE of Fluorophore-labeled Saccharides) or in one
commercial format as FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). In the PAGEFS method, saccharides having an
aldehydic reducing end group are labeled quantitatively with a fluorophore and then separated with high resolution by PAGE.
Two fluorophores, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS) and 2-aminoacridone (AMAC), have been used to enable the
separation of a variety of saccharide positional isomers, anomers, and epimers. Subpicomolar quantities of individual saccharides
can be detected using a sensitive imaging system. Mixtures of oligosaccharides obtained by enzymatic cleavage from glycoproteins
can be labeled and electrophoresed to yield an oligosaccharide profile of each protein. AMAC can be used to distinguish unequivocally
between acidic and neutral oligosaccharides. Methods for obtaining saccharide sequence information from purified oligosaccharides
have been developed using enzymatic degradation. Other applications and the potential of the system are described. |
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Keywords: | Carbohydrates saccharides glycans electrophoresis polyacrylamide fluorophore-labeling PAGEFS FACE CCD-imaging |
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