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The relatively new approach of lectin affinity chromatography has become a standard and widely used technique for the isolation of soluble glycoproteins, hormones, antigens and polysaccharides, as well as detergent-solubilized membrane-bound glycoconjugates and cell surface receptors. An excellent review article on this topic has been published by Lotan and Nicolson (Biochim. Biophys. Acta, 559, 329-376, 1979).
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Lectin affinity chromatography combines simplicity with potentially high resolution. During the purification of glycoconjugates, the immobilized lectin is allowed to bind to the glycoconjugate, and the unbound residual material can be readily removed by subsequent washing. The bound glycoconjugates are displaced from the immobilized lectins by the addition of a solution of a sugar known to inhibit binding of the particular lectin.
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A continually increasing number of glycoproteins have been purified by using immobilized lectins. Among these are hormones, blood group substances, viral glycoproteins, histocompatibility antigens, interferons, enzymes, lymphocyte markers, serum proteins, and oncofetal antigens.
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Our immobilized lectins are prepared from affinity-purified lectins. Heat stable, cross-linked 4% agarose beads with a molecular weight exclusion limit of about 2x107 are used as the solid-phase matrix to which the lectins are covalently bound. The attachment of the lectins to the solid phase is carefully controlled in order to preserve the activity of the lectins as well as to minimize conformational changes of the bound lectins which might result in nonspecific ionic or hydrophobic interactions.
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The technique we have developed to couple lectins to agarose provides a very hydrophilic spacer arm between the protein and the matrix. This ensures maximum expression of the carbohydrate binding activity of the lectin. The linkage is very stable over a range of pH values and, unlike cyanogen bromide linkages, proteins are not leached off the gel by Tris or other routinely used buffers. In addition, residual charges generated during cyanogen bromide conjugation which can produce nonspecific binding are not present on the gel following our coupling procedure.
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Our agarose bound lectins are supplied at a constant concentration of lectin per ml of gel. The concentration at which each lectin is prepared is selected in order to achieve the highest glycoconjugate binding capacity per mg of lectin present in the gel.
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Before each lot is made available, its binding capacity is tested using glycoproteins or derivatives of glycoproteins known to bind to the particular lectin being evaluated. This provides a guideline for the user and assures the quality of our agarose bound lectins.
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