Affinity chromatography ligand coupling

A third item to consider in using affinity chromatography is the way in which the ligand is attached to the solid support, or the immobilization method. Several techniques are available for this, including both covalent and noncovalent coupling methods [25,36]. For a protein or peptide, this generally... 

Table 13.3 summarizes various covalent immobilization methods that are used in affinity chromatography. Each of these methods involves at least two steps (1) an activation step, in which the support is converted to a form that can be chemically attached to the ligand and (2) a coupling step, in which the affinity ligand is attached to the activated support. With some techniques, a third step, in which remaining activated groups are removed, is also required. The methods listed in Table 13.3 can be performed either in-house or can be used in the form of preactivated supports available from commercial suppliers (see list in Table 13.2) [25,36]. 

Many other means of preparation of adsorbents for affinity chromatography are also available.121122 For example, l,l -carbonyl-diimidazole can be used to couple a diamine to the matrix (Fig. 3-11). This reagent has the advantage that it does not depend upon the relatively unstable isourea linkages formed by Eq. 3-9 to hold the specific affinity ligands.121... 

The methods for production of such immobilized ligands and for carrying out affinity purification of IgG are essentially similar whatever ligand is used. Sepharose 4B is probably the most widely used matrix for affinity chromatography, but there are other materials available. Activation of Sepharose 4B is usually carried out by reaction with CNBr this can be carried out in the laboratory before coupling or ready-activated lyophihzed Sepharose can be purchased. The commercial product is obviously more convenient than homemade activated Sepharose, but it is more expensive and may be less active. 

The basis for selectivity in affinity chromatography is the use of immobilized biochemicals, known as affinity ligands, that are covalently attached to a support matrix, as illustrated in Figure 2.17. The primary criteria that govern the suitability of a support matrix for affinity chromatography include (1) the mechanical and flow properties of the matrix, (2) the ease of covalent coupling of the ligand to the matrix, and (3) the stability of the... 

A two-column affinity chromatography method, Fig. (20 ), was devised and used to separate the anti-arabic antibodies into two sets by using two types of adsorbents [56], For the preparation of one adsorbent a sample of gum arabic was hydrolyzed in 0.01 M HC1 for 10 min and the acid hydrolyzed gum was coupled to AH-Sepharose 4B. The second adsorbent was synthesized by coupling native gum arabic to AH-Sepharose 4B. The columns were attached in a series with the hydrolyzed gum arabic ligand-column preceding the native gum arabic ligand-column. In order to decrease... 

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