Erythrina cristagalli lectin (ECL, ECA), Agarose bound

Erythrina cristagalli lectin consists of two different subunits of approximately 28 kDa and 26 kDa. The carbohydrate structure to which ECL binds is frequently found in membrane and serum glycoproteins of mammalian origin. Sialic acid substitution on this structure appears to prevent the lectin from binding. This specificity offers an opportunity to utilize agarose bound ECL to isolate or fractionate mammalian glycoproteins. This lectin has been reported to be useful for the isolation of human natural killer (NK) cells using a negative selection panning technique (protocol available upon request or on our website). Human NK cells appear to lack accessible surface carbohydrate structures required for binding ECL and, unlike other mononuclear cells, do not adhere to ECL-coated culture dishes. Since this procedure involves a negative selection panning technique, a high recovery of viable NK cells can be obtained. The adherent cells can also be recovered by incubation in galactose or lactose. Agarose bound* Erythrina cristagalli lectin is 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. Inhibiting/Eluting Sugar: 200 mM lactose. *3 mg lectin/ml gel.

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