Polyacrylamide Gel Electrophoresis of Fluorophore-Labelled Reducing Saccharides: A Review

Abstract

Polyacrylamide gel electrophoresis (PAGE) is the standard method for the analytical separation of proteins and for DNA sequencing. In contrast, PAGE has been little used for carbohydrates, and a wide variety of other separation methods are in common use, such as thin-layer chromatography (1), gel permeation chromatography using either soft gels (2) or various forms of high pressure liquid chromatography (HPLC) (3,4) and capillary electrophoresis (5). However, some workers have applied PAGE for saccharide separations; for instance, Gallagher and co-workers (6,7) separated glycosaminoglycans (see Chapter 13), and Das and Henderson (8) demonstrated the separation of oligosaccharides by PAGE. One possible reason for the lack of application of PAGE in this field is that it is usually used to separate relatively large molecules and requires adaptation for high-resolution separations of small molecules such as those of particular interest to glycobiologists. Moreover, small molecules can present problems in detection when employing post-electrophoretic staining methods, and it is common to label such substances before electrophoresis with either a suitable dye or fluorophore or radioisotope. By incorporating a pre-separation fluorophore-labelling strategy, PAGE can now be adapted for the analysis of reducing saccharides. This method, polyacrylamide gel electrophoresis of fluorophore-labelled saccharides (PAGEFS) [also called fluorophore-assisted carbohydrate electrophoresis (FACE) in its commercial format], described in this chapter, is a novel addition to carbohydrate separation techniques in which saccharides that have a reducing terminal (aldose) are separated by PAGE after derivatization by a suitable fluorophore (9–24).