Affinity chromatography steps involved

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]. 

Purification of a recombinant therapeutic protein from cell culture may involve a few simple steps. For example, affinity chromatography may be used to concentrate the protein. This can be followed with an ion-exchange step and gel filtration. 

These property-oriented discovery technologies are specifically supramolecular since they are based on interactions between species. They present common features with procedures such as affinity chromatography, vaccine discovery and isolation by immunoprecipitation or induction of microbiological resistance. In their full expression, they involve three main steps diversity generation, effector selection and identification through encoding. 

While there are no set protocols for the specific order in which the different chromatographic separations are carried out, ion exchange and hydrophobic interaction chromatography typically precede gel filtration and bio-affinity chromatography. The latter can involve expensive resins and is often performed after various contaminants have been removed during earlier purification steps. 

Acetyl-CoA arylamine N-acetyltransferase was purified from pigeon liver by using steps involving protamine sulfate, ammonium sulfate fractionation, and affinity chromatography on immobilized amethopterin. Figure 9.20 shows a representative chromatogram. In another study, Thomas et al. (1990) used tryptamine as the substrate. In addition, whereas the study by Manneus et al. (1990) appeared to require pure enzyme, Thomas s study did not. 

As an example of the steps involved in affinity chromatography, we will consider the purification of an enzyme present in a mixture of proteins, using an immobilized competitive inhibitor as an affinity ligand. The first step after column preparation is the application of the sample during this step, the enzyme that binds to the inhibitor is retained, or adsorbed on the column. This is followed by a rinse step, whereby all nonbinding species are removed. The third step involves elution, and this step... 

A comprehensive proteomics approach has been developed to identify the components of phosphoprotein complexes and includes the following experimental steps isolation of native phosphoproteins and associated proteins by affinity chromatography, ID PAGE separation of the affinity-isolated proteins, proteolysis of the protein spots, LC-ESI-MS/MS analysis of the cleaved peptides, and database searching for indentification of the proteins involved in the complex formation.178... 

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