Immobilized Metal Affinity Chromatography (IMAC)
Supports such as beaded agarose or magnetic particles can be derivatized with chelating groups to immobilize the desired metal ions, which then function as ligands for binding and purification of biomolecules of interest. This basis for affinity purification is known as immobilized metal affinity chromatography (IMAC).
The chelators most commonly used as ligands for IMAC are nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). Once IDA-agarose or NTA-agarose resin is prepared, it can be "loaded" with the desired divalent metal (e.g., Ni, Co, Cu, Fe). Using nickel as the example metal, the resulting affinity support is usually called Ni-chelate, Ni-IDA or Ni-NTA resin.
The particular metal and chelation chemistry of a support determine its binding properties and suitability for specific applications of IMAC. Affinity purification of His-tagged fusion proteins is the most common application for metal-chelate supports in protein biology research. Nickel or cobalt metals immobilized by NTA-chelation chemistry are the systems of choice for this application (see next section).
In addition, different varieties of agarose resin provide supports that are ideal for His-tagged protein purification at very small scales (96-well filter plates) or large scales (series of chromatography cartridges in an FPLC system). When packed into suitable columns or cartridges, resins such as Ni-NTA Superflow Agarose provide for purification of 1 to 80 milligrams of His-tagged protein per milliliter of agarose beads.
Poly-His tags bind best to IMAC resins in near-neutral buffer conditions (physiologic pH and ionic strength). A typical binding/wash buffer consists of Tris-buffer saline (TBS) pH 7.2, containing 10-25mM imidazole. The low-concentration of imidazole helps to prevent nonspecific binding of endogenous proteins that have histidine clusters. (In fact, antibodies have such histidine-rich clusters and can be purified using a variation of IMAC chemistry.)
High concentrations of salt and certain denaturants (e.g., chaotropes such as 8M urea) are compatible, so purification from samples in various starting buffers is possible. For this reason, it is best to use the His tag for design and expression of recombinant proteins that may need to be purified in denatured form from inclusion bodies. (Contrast this with the GST tag, which is an enzyme that must remain functional to enable purification.) However, reducing agents, oxidizing agents and chelators (e.g., EDTA) are generally not compatible with IMAC affinity chemistry.
Elution and recovery of captured His-tagged protein from an IMAC column is accomplished with a high concentration of imidazole (at least 200mM), low pH (e.g., 0.1M glycine-HCl, pH 2.5) or an excess of strong chelator (e.g., EDTA). Imidazole is the most common elution agent.
Be aware that immunoglobulins are known to have multiple histidines in their Fc region and can bind to IMAC supports. High background and false positives can result if binding conditions are not sufficiently stringent (i.e., with imidazole) and the immunoglobulins are abundant relative to the His-tagged proteins of interest. Albumins, such as bovine serum albumin (BSA), also have multiple histidines and can bind to the IMAC supports in the absence of His-tagged proteins in the sample or imidazole in the binding/wash buffer.