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Matrices for Glycobiology

Lectin affinity chromatography is a simple and widely used technique for the isolation and purification of a variety of glycoproteins and other glycoconjugates. The glycoconjugate is allowed to bind to the agarose bound lectin, and the unbound residual material is removed by washing. The bound glycoconjugates are generally eluted with a solution of a sugar known to inhibit binding of the particular lectin. Soluble glycoproteins, hormones, antigens, polysaccharides, detergent-solubilized membrane-bound glycoconjugates, cell surface receptors, blood group substances, viral glycoproteins, histocompatibility antigens, lymphokines, enzymes, lymphocyte markers, serum proteins, and oncofetal antigens are only a few of the substances that have been purified using lectins matrices.

Our agarose bound lectins are prepared using our affinity-purified lectins. Heat stable, cross-linked 4% agarose beads with a molecular weight exclusion limit of about 2x107 daltons are used as the solid-phase matrix to which the lectins are covalently coupled. The attachment of the lectins to the beads is carefully controlled to preserve lectin activity and minimize conformational changes of the bound lectins that might result in nonspecific ionic or hydrophobic interactions. The technique we have developed to couple lectins to agarose beads inserts a hydrophilic spacer arm between the lectin and the matrix.

This coupling method provides several advantages over the traditional cyanogen bromide procedure:

  • - Maximum carbohydrate binding activity of the coupled lectins is retained
  • - Linkage is stable over a range of pH values
  • - Conjugated proteins are not leached off the beads by Tris or other routinely used buffers
  • - No residual charges are present after conjugation. This minimizes non-specific binding to the matrix.

Our agarose bound lectins are supplied at a constant concentration of lectin per ml of settled beads. The concentration for each lectin is selected to achieve the highest glycoconjugate binding capacity per mg of lectin present in the beads. Each lot is tested for its binding capacity using glycoproteins known to bind the lectin. This provides a guideline for the user and assures the quality of our agarose bound lectins.