Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The degree of purification can be quite high depending on the specificity of the interaction and, consequently, it is generally the first step, if not the only step, in a purification strategy.
This section provides an overview of affinity chromatography of proteins, general considerations for affinity purification of proteins, and provides some commonly used affinity chromatography methods.
Related Topics: Size Exclusion Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction Chromatography, Mixed-Mode Chromatography, Low Pressure Chromatography Systems and Medium Pressure Chromatography Systems.
Affinity chromatography offers high selectivity, resolution, and capacity in most protein purification schemes. It has the advantage of utilizing a protein's biological structure or function for purification. As a result, purifications that would otherwise be time consuming and complicated, can often be easily achieved with affinity chromatography.
A commonly used metaphor to illustrate affinity binding is the lock and key analogy. A unique structure present on the surface of a protein is the key that will only bind to the corresponding lock, a specific ligand on a chromatographic support.
In two-step affinity-tagged protein purification, a protein is first purified by affinity chromatography, then desalted. In some medium pressure chromatography systems, such as the NGC medium pressure chromatography systems, these two steps can be automated. In the first step, a recombinant protein mixture is passed over a chromatography support containing a ligand that selectively binds proteins that contain an affinity-tag sequence (typically His or GST). Contaminants are washed away, and the bound protein is then eluted in pure form.
Affinity tags have different advantages. In immobilized metal affinity chromatography (IMAC), His binds with good selectivity to Ni2+ or other transition metals immobilized to the ligand; the tagged protein can be selectively eluted with imidazole. proteins tagged with GST bind to glutathione as the ligand, and are eluted with solutions of glutathione. Proteins with an enzymatically active GST fusion tag can only be purified under native conditions. In contrast, polyhistidine-tagged proteins may be purified under native or denaturing conditions.
During the second step of desalting, affinity-purified samples can simultaneously undergo buffer exchange to remove salts in preparation for downstream applications.
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.
Affinity chromatography can be broadly divided into two method types:
Bio-Rad offers ready to use affinity media and customizable or activated media.
Ready to Use Affinity Media Selection Guide
Activated media: Affinity chromatography can also include customized media. For instance, a specific ligand can be attached to an activated or coupled resin. Samples containing proteins that will bind to this ligand will be retained. One example is the attachment of DNA to beads via a coupling linkage. DNA-binding proteins (such as polymerases) will be retained on the media.
* Refer to bulletin 3193 for purification conditions. ** For example, Profinity GST resin is only available in prepacked cartridges. *** Profinity eXact purification resin.
The binding and elution conditions in affinity chromatography can vary greatly, however, there are some general guidelines. Because most binding interactions are based on those formed in nature, the conditions similar to those present in most cellular organisms are usually the best binding conditions. Therefore PBS (phosphate buffered saline) is often the buffer of choice. Conversely, conditions not normally found in vivo may alter the protein structure enough to cause the protein to dissociate from the ligand. In situations where there is no recommended elution condition, a low pH (>4) will often elute bound protein with an inherent risk of denaturation.
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