Blotting and Detecting Glycosaminoglycans

Glycosaminoglycans (GAGs) are non-branched (linear), complex polysaccharides containing a variable number of repeating disaccharide units (hence called heteropolysaccharides). GAG separation and characterization by electrophoresis are an important part of biochemical research. Immunoblotting of GAGs after electrophoresis helps carry out additional analysis on single, separated species, such as identifying with antibodies and/or recovery of a single polysaccharide type. Nicola Volpi and Francesca Maccari developed a technique for the blotting (after regular agarose gel electrophoresis) and immobilizing of several non-sulfated and sulfated GAGs on membranes that have been made hydrophilic and positively charged by cationic detergent. A mixture of GAGs are capillary blotted, following agarose gel electrophoresis, nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride. Distinct purified types of sulfated polysaccharides are transferred to these modified membranes with 100% efficiency and stained with Alcian blue (irreversible staining) and toluidine blue (reversible staining). This permits a higher sensitivity of detection, detecting as low as 0.1 μg. Hyaluronic acid (an example of non-sulfated polyanions) can also be blotted to membranes (detection limit of about 0.1–0.5 μg) after irreversible or reversible staining. The membranes can also be stained with reversible staining and the same membrane used for immunological detection or other applications.