Affinity chromatography covalent immobilization method

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]. 

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... 

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

The protein adsorption on active carbons is an important process contributing to their biocompatibility and the therapeutic effect of hemoperfusion (Arshady 1999). Covalent binding of proteins to a carbon surface allows one to synthesize adsorbents with high selectivity of action similar to that achieved in affinity chromatography. Despite numerous studies of carbon adsorbent interactions with biomolecules in aqueous media, the influence of the adsorbent structure on the characteristics of the interfacial layer of water in the presence of immobilized proteins ranains unclear. Here the relationships between the structural characteristics of activated carbon SCN and the state of the bound water in the presence of BSA and mouse y-globulin (IgG) physically adsorbed or covalently attached to the carbon surface via carbodiimide coupling are analyzed using adsorption and H NMR methods. 

Technologies for immobilizing proteins on surfaces that have been developed over the last 25 years are particularly critical to the continued development of biosensors. There a dozens of techniques for activating surfaces so as to be able to covalently bond proteins while maintaining their biological activity. These methods have been used routinely for manufacturing adsorbents for affinity chromatography, dip-stick type analytical methods, e.g., immunoassays, and immobilized enzymes for commercial catalysts. 

---