Affinity ligands Matrix coupling

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 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 widely applicable, non-chaotropic elution technique in bioaffinity chromatography is electrophoretic desorption [60,74,75]. If the affinant is coupled to the solid support by an azo bond or by thiol- or alcohol-ester bonds, the complex of the affinant with the isolated substance can be detached from the solid matrix and then the affinity ligand separated by dialysis or gel filtration. 

Affinity chromatography can fail for reasons other than inappropriate ligand. You need to watch out for the length and type (hydrophile/hydrophobic) of the spacers, the chemical nature and pore size of the matrix, and the buffer (ion types, pH, detergent, ion strength) in which the sought-after protein is offered to the matrix. Moreover, ligands rarely couple as well and as irreversibly as the vendor brochures claim. 

On the other hand, ready-to-use matrices have a specific ligand already coupled to an affinity support. Amersham Pharmacia Biotech has developed a wide range of ready-to-use affinity gels with specific protein ligands coupled to the matrix. In specified bulletins, Amersham Pharmacia... 

One of the most important factors in the area of affinity chromatography is the development of solid supports. A correct choice of a solid support and the covalent coupling between the matrix and the affinity ligand may be essential for the success of the desired separation. A solid support may even constitute an affinity ligand itself, for example, polysaccharides for some lectins [63]. 

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. 

---