• Affinity Chromatography

    Posted on Nov 15, 2018

    Purification of a protein is accomplished by the initial binding of the protein to an immunogen, in the case of an antibody, or to a substrate in the case of an enzyme. Other affinity purifications could be an immobilized DNA for a DNA-binding protein, immobilized ligand for a receptor-ligand pair or an immobilized ligand that binds a tag on a recombinant protein. Affinity columns can be made by covalently crosslinking the ligand to the support gel, such as Sepharose. The bound protein is separated from contaminating proteins by washing the column thus eluting contaminants, and subsequently eluting the protein of interest by acid or chaotropic reagents followed by recovery of activity. Typically, good separations of the protein from contaminants are achieved so that only one or two subsequent purification methods will resolve the protein to nearly homogeneity.

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