Glycosaminoglycan Blotting and Detection After Electrophoresis Separation

Separation of glycosaminoglycans (GAGs) by electrophoresis
and their characterization to the microgram level are integral
parts of biochemical research. Their blotting on membranes after
electrophoresis offers the advantage to perform further analysis on
single separated species such as identification with antibodies and/or
recovery of single band. A method for the blotting and immobilizing of
several nonsulfated and sulfated complex GAGs on membranes made
hydrophilic and positively charged by cationic detergent after their
separation by conventional agarose-gel electrophoresis is illustrated.
This approach to the study of these complex macromolecules utilizes
the capacity of agarose-gel electrophoresis to separate single species
of polysaccharides from mixtures and the membrane technology for
further preparative and analytical uses.
Nitrocellulose membranes are derivatized with the cationic detergent
cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary
blotted after their separation in agarose-gel electrophoresis. Single
purified species of variously sulfated polysaccharides are transferred
on derivatized membranes with an efficiency of 100 % and stained
with alcian blue (irreversible staining) and toluidine blue (reversible
staining). This enables a lower amount limit of detection of 0.1 μg.
Nonsulfated polyanions, for example hyaluronic acid (HA), may
also be transferred to membranes with a limit of detection of
approximately 0.1–0.5 μg after irreversible or reversible staining.
The membranes may be stained with reversible staining and the
same lanes used for immunological detection or other applications.