Affinity chromatography is a method of separation that uses binding interaction specific to a ligand immobilized in a chromatographic column and its molecular binding partner. This method may use antibody and antigen, enzyme and substrate, or enzyme and inhibitor. Affinity chromatography can achieve a high degree of purification, depending on the specificity of the interaction. It is so efficient that it is generally the first step or the only step in a purification strategy.
Why use affinity chromatography?
Affinity chromatography can be scaled up for the purification of large amounts of substrate. It is highly selective at high resolution. The advantages of affinity chromatography derive from the fact that it uses a protein’s biological function or structure or purification. Purifications that otherwise would be prohibitively time-consuming and complicated can be easily achieved with affinity chromatography.
How does affinity chromatography work?
Affinity chromatography uses the principle of affinity binding, often explained in terms o the example of a lock and key. The unique surface that appears on the surface of a protein is analogous to a key that will only bond with a specific ligand on chromatographic support.
How does affinity chromatography provide a purified product?
Affinity-tagged protein purification is a two-step process. First, the protein is purified by affinity chromatography. Then, it is desalted.
In commercial affinity chromatography purification processes, such as the NGC medium-pressure chromatography systems, these two steps are automated. In the first step, a recombinant protein mixture passes over a chromatography column containing a ligand that selectively binds to proteins that contain a ligand that contains an affinity-tag sequence. The system washes contaminants away, eluting the bound protein in pure form
Affinity tags have several advantages. In immobilized metal affinity chromatography, histidine binds selectively to nickel ions or other transition metals immobilized to the ligand. Tagged proteins can be eluted selectively with imidazole. Proteins tagged with glutathione S-transferase bind to glutathione as the ligand and can be eluted with glutathione solutions. Proteins with an enzymatically active glutathione S-transferase fusion tag cannot be purified under denaturing conditions. Proteins tagged with polyhistidine can be purified under either denaturing or native conditions.
In the second step of desalting, affinity-purified samples can be sent through a buffer exchange to remove salts to prepare them for downstream applications.
Buffer exchange is made possible by a number of desalting techniques, including size exclusion chromatography, ultrafiltration, and dialysis. Desalting usually removes not only salts, but also other foreign substances, such as nucleotides, lipids, and detergents.
A number of desalting techniques, including size exclusion chromatography, dialysis, and ultrafiltration, also allow buffer exchange. Desalting often includes the removal not only of salt, but also of other foreign substances, such as detergents, nucleotides, and lipids.
What are the two types of affinity chromatography?
There are two methods of affinity chromatography:
- The first method utilizes a naturally occurring sequence of amino acids or a naturally occurring structure on the protein as the binding site. Examples of this method include Affi-Gel Blue support binding for the bilirubin binding site of albumin. The binding of protein A in the Affi-Prep and Affi-Gel protein A supports the Fc region of IgG. To use this method, operators must determine the affinity of their target antibody for their protein A/G chromatography media.
- The second common method of affinity chromatography binds a known amino acid sequence that has been engineered into the target of interest. This amino acid sequence is commonly called a “tag.” Two commonly used tags are the polyhistidine tag, which bonds to metal-containing complexes, and
- The second method uses binding to a known amino acid sequence that has been engineered into the protein of interest. This amino acid sequence is commonly known as the “tag.” A number of different tags are available. Two of the most commonly used protein tags are the polyhistidine tag, which binds to certain metal-containing complexes, and the GST (glutathione S-transferase) sequence, binding to glutathione. In theory, any protein can be purified by a tagging method, although there are many factors to consider in designing a process to purify tagged recombinant proteins.