Immobilization of Proteoglycans Glycosaminoclycan Chains and Oligosaccharides

The immobilization of ligands is accomplished by chemically activating a functional group on the surface, e.g., carboxyl on carboxymethyl dextran or amino on aminosilane. Since excellent protocols for ligand immobilization are supplied by the manufacturers, only points specific to proteoglycans will be considered here. There are major difficulties in the efficient direct coupling of proteoglycans, glycosaminogly-can chains, and oligosaccharides to surfaces, due to the highly anionic character of these molecules, which is a consequence of the carboxyl and sulfate groups present on the saccharides. Thus, electrostatic uptake strategies, which are used to concentrate ligand on carboxymethyl dextran surfaces to increase the efficiency of immobilization, will not work, since the isoelectric point of the polysaccharide chains is lower than that of the carboxyl groups on the matrix. Moreover, due to the high negative

Binding Dextran Surface Cells

Fig. 2. Identification of zero background surfaces. Ligate is added in the buffer chosen for binding assays at the highest concentration likely to be used in these assays. The most commonly used detergent is Tween-20. In 0.5% (v/v) Tween-20 no background binding is observed with crude lysates of tissues or cells on streptavidin-derivatized IAsys aminosilane surfaces (Wain-wright, G. and Fernig, D.G., unpublished observations). Changing the charge of the surface at physiological pH, e.g., negative charge of carboxymethyl dextran, is accomplished by repeated activation of the charged groups of the surface. An elegant example is provided in ref. (8).

Fig. 2. Identification of zero background surfaces. Ligate is added in the buffer chosen for binding assays at the highest concentration likely to be used in these assays. The most commonly used detergent is Tween-20. In 0.5% (v/v) Tween-20 no background binding is observed with crude lysates of tissues or cells on streptavidin-derivatized IAsys aminosilane surfaces (Wain-wright, G. and Fernig, D.G., unpublished observations). Changing the charge of the surface at physiological pH, e.g., negative charge of carboxymethyl dextran, is accomplished by repeated activation of the charged groups of the surface. An elegant example is provided in ref. (8).

charge of the glycosaminoglycan chains, proteoglycans and their saccharide components will not penetrate readily into the carboxymethyl dextran gel in the absence of a counterion. To obtain reasonable levels of immobilized ligand in the absence of electrostatic uptake requires a high concentration of ligand (>1 mg/mL) during the coupling reaction. Samples of proteoglycan, glycosaminoglycan chains, and oligosaccharides are usually fairly precious. A capture system such as biotin-streptavidin avoids the problems inherent to the direct coupling of proteoglycans and derived polysaccharides, and so is the preferred method for the immobilization of these ligands. In addition, the capture of biotinylated proteoglycans and polysaccharides allows the oriented immobilization of the ligand, which optimizes ligate binding (see Subheading 3.5.5.).

Proteoglycans and glycosaminoglycan chains isolated as peptidoglycans can be conveniently biotinylated on amino groups. Free amino groups may also occur along the polysaccharide chain. If required, the relative level of biotinylation of peptide and polysaccharide can be estimated, with respect to a specific ligate, e.g., basic fibroblast growth factor (bFGF) (3). Oligosaccharides produced by enzymatic cleavage of glycosaminoglycan chains, e.g., heparinase, will contain a reducing end. Again this provides a unique functionality that can readily be biotinylated.

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