Chromatography, affinity covalent

Figure 4.5. Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G). 

Purified by (NH4)2S04 fractionation, followed by PC cellulose chromatography and affinity chromatography (using Sepharose 4B to which (G)n was covalently bonded). [Schmukler et al. J Biol Chem 250 2206 7975.]... 

Candidates for the renal brush border Na /H exchanger transport protein identified by covalent labeling, affinity chromatography, or other methods... 

Compared with enzymes fewer reports are available on immobilization of antibody (Ab) in sol-gels and their applications in immunosensing. Immobilization of Abs on a solid support was first reported in 1967 [128] and the technology has widespread application in affinity chromatography and other areas. However, the major problem associated with covalent immobilization of antibody on solid surface is partial loss of biological activity due to the random orientation of the asymmetric macromolecules,... 

The possible formation of a dipole is a feature of covalent bonding but it is obvious that an ionic bond results in a definite unequal distribution of electrons within a molecule and such molecules (or ions) are extremely polar. However, the fact that they carry a definite charge enables additional separation techniques to be applied. The rate of migration in an electric field (electrophoresis) and the affinity for ions of opposite charge (ion-exchange chromatography) are extremely valuable techniques in the separation of ionic species. 

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]. 

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