Binding and Detection of Glycosaminoglycans Immobilized on Membranes Treated with Cationic Detergents

Abstract

Immobilization of molecules on surfaces is used for preparative, quantitative, and qualitative studies. Glycosaminoglycans (GAGs) are strongly hydrophilic and negatively charged molecules that do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. Hydrophobic membranes were derivatized with cationic detergents to become hydrophilic and positively charged. The ability of the polyvinylidene fluoride and nitrocellulose membranes to retain GAGs increased up to 12.8 μg per spot in the dot blot assay when the membrane was treated with a cationic detergent. Immobilized GAGs were stained with alcian blue, and the staining intensity was quantitated by scanning and densitometry. The derivatized membranes were used for solid-phase extraction of GAGs in blood plasma, urine, or cerebrospinal fluid. The detection sensitivity was equal for different types of GAGs but there was a slight negative interference from fibrinogen in blood plasma. The immobilized GAGs could also be released from the membrane using a nonionic detergent at high ionic strength. Recovery of different proteoglycan populations, separated by electrophoresis and detected by reversible staining with toluidine blue, was 70–100%.