![]() ![]() The resin provides a high degree of purity and may recover more than 10 mg of pure His-tagged protein per milliliter of resin without metal contamination or the need to optimize imidazole washing conditions.Enzyme catalysis has been studied for over a century. Thermo Scientific HisPur Cobalt Resin is a tetradentate chelating agarose resin charged with divalent cobalt (Co 2+). Albumins, such as bovine serum albumin (BSA), also have multiple histidines and can bind to IMAC supports in the absence of His-tagged proteins in the sample or imidazole in the binding/wash buffer. 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. Imidazole is the most common elution agent.īe aware that immunoglobulins are known to have multiple histidines in their Fc region and can bind to IMAC supports. They are specifically suited for purifying expressed His-tagged proteins that are secreted into cell culture media, or for purifying intracellular His-tagged proteins that need the presence of EDTA to maintain stability and function.Įlution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). ![]() The EDTA compatible Ni-IMAC chemistry is available in magnetic bead (Cat. But a specially engineered Ni-IMAC chemistry is available that can tolerate the presence of reducing agents and chelators such as EDTA at higher concentrations without the loss of performance. (Contrast this with the GST-tag, which is an enzyme that must remain functional to enable purification.) It is important to note that EDTA and reducing agents such as DTT and TCEP can adversely affect the performance of regular Ni-IMAC supports by stripping off the metal. 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. High concentrations of salt and certain denaturants (e.g., chaotropes such as 8 M urea) are compatible, so purification from samples in various starting buffers is possible. (In fact, antibodies have such histidine-rich clusters and can be purified using a variation of IMAC chemistry.) The low-concentration of imidazole helps to prevent nonspecific binding of endogenous proteins that have histidine clusters. A typical binding/wash buffer consists of Tris-buffer saline (TBS) pH 7.2, containing 10-25 mM imidazole. Poly-His tags bind best to IMAC resins in near-neutral buffer conditions (physiologic pH and ionic strength). Thermo Fisher Scientific offers HisPur Ni-NTA Superflow Agarose that exhibits a high dynamic binding capacity across a range of flow rates, making it an excellent choice for larger scale purifications. Compared to cobalt and other ligands used for IMAC, nickel provides greater capacity for His-tagged protein purification. ![]() 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. 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). Nickel or cobalt metals immobilized by NTA-chelation chemistry are the systems of choice for this application (see next section). The particular metal and chelation chemistry of a support determine its binding properties and suitability for specific applications of IMAC.Īffinity purification of His-tagged fusion proteins is the most common application for metal-chelate supports in protein biology research. Using nickel as the example metal, the resulting affinity support is usually called Ni-chelate, Ni-IDA or Ni-NTA resin. Once IDA-agarose or NTA-agarose resin is prepared, it can be "loaded" with the desired divalent metal (e.g., Ni, Co, Cu, and Fe). The chelators most commonly used as ligands for IMAC are nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). IMAC is a widely-used method for rapidly purifying polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purification step. This basis for affinity purification is known as 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.
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