Affinity chromatography ligand attachment

Sundberg, L., and Porath, J. (1974) Preparation of adsorbents for biospecific affinity chromatography. I. Attachment of group containing ligands to insoluble polymers by means of bufunctional oxiranes. /. Chromatogr. 90, 87—98. 

Affinity Chromatography The ability of proteins to bind specifically to other molecules is the basis of affinity chromatography. In this technique, ligand molecules that bind to the protein of interest are covalently attached to the beads used to form the column. Ligands can be enzyme substrates or other small molecules that bind to specific proteins. In a widely used form of this technique, antibody-affinity chromatography, the attached ligand is an antibody specific for the desired protein (Figure 3-34c). 

Figure 6.14 Schematic representation of the principle of biospecific affinity chromatography. The chosen affinity ligand is chemically attached to the support matrix (agarose bead) via a suitable spacer arm. Only those ligands in solution that exhibit biospecific affinity for the immobilized species will be retained...

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]. 

FIGURE 13.4 The effects of (a) multisite attachment, (b) random or improper orientation, and (c) steric hindrance on an immobilized ligand. (Reproduced from Kim, H.S. and Hage, D.S., Immobilization methods for affinity chromatography, in Handbook of Affinity Chromatography, 2nd edn., Hage, D.S. (ed.), CRC Press, Boca Raton, FL, 2005, Chap. 3.)... 

Affinity chromatography is a very powerful technique which separates biomolecules according to differences in their biological function or chemical stracture. The stationary phase for affinity chromatography consists of a matrix to which ligands are covalently attached. 

Selection of the matrix used to immobilize a ligand requires consideration of several properties. The stationary supports used in gel exclusion chromatography are found to be quite suitable for affinity chromatography because (1) they are physically and chemically stable under most experimental conditions, (2) they are relatively free of nonspecific adsorption effects, (3) they have satisfactory flow characteristics, (4) they are available with very large pore sizes, and (5) they have reactive functional groups to which an appropriate ligand may be attached. 

Affinity chromatography exploits the specific, high affinity, noncovalent binding of a protein to another molecule, the ligand. First, the ligand is covalently attached to an inert and porous matrix (such as Sepharose). The protein mixture is then... 

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... 

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