Due to its relatively small size, low immunogenicity, hydrophilic nature and versatility in the presence of detergents and many other additives, and under native and denaturing conditions, the polyhistidine tag is now considered as the most widely used affinity tag for a variety of protein purification purposes.
In addition, its ability to detect and purify recombinant proteins without using a protein-specific antibody or probe, and the availability of anti-His tag antibodies for use in assays involving His-tagged proteins make 6xHis tags all the more popular.
Affinity Purification of Proteins
Polyhistidine-tagged recombinant proteins expressed in E. coli and other prokaryotic systems are usually purified using immobilized metal ion affinity chromatography or IMAC. Under this process, the support (usually a beaded agarose or magnetic particles) is derivatized with the appropriate chelating agent such as nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).
These chelating groups will immobilize the desired divalent metal ions (such as nickel, copper, cobalt and/or zinc) which will, in turn, be responsible for binding and separating the biomolecule of interest from a mixture of proteins. In choosing which ligand to use, you need to consider the fact that the binding capacity of an IMAC resin is heavily dependent on the nature of the protein being purified and the metal ion used in the process.
Nickel, cobalt and copper are commonly used in the purification of His-tagged proteins primarily due to their affinity to histidine and cysteine in aqueous solutions. Among the three, nickel is, by far, the most widely available metal ion and exhibits the highest affinity and selectivity to His-tagged proteins. However, it tends to bind nonspecifically to endogenous proteins containing histidine clusters.
On the other hand, cobalt provides more specific binding with histidine tags so it becomes the divalent cation of choice when purity is a primary concern.
Copper ions bind His-Tags more strongly as compared to both nickel and cobalt but it provides the lowest specificity among the three. Due to this reason, copper IMAC is only used when purity is not the primary objective.
IMAC Methods
In preparing the sample, the cells are harvested and lysed by enzymatic or mechanical conditions. Since poly-His tags bind best to IMAC resins at physiologic pH and ionic strength, binding is done at near neutral buffer conditions. Most researchers use Tris-buffer saline (TBS) containing 10 to 25mM imidazole as binding/wash buffer to prevent nonspecific binding of endogenous proteins with histidine clusters. Elution and recovery of captured His-tagged proteins is usually done using increased imidazole concentrations (at least 200 mM), low pH or an excess of a strong chelators such as EDTA.
You can purify samples in various starting buffers since high concentrations of salt and certain denaturants are compatible. However, please take note that reducing agents, oxidizing agents and chelators such as EDTA are generally not compatible with IMAC affinity chemistry.
Other Applications for His-Tagged Proteins
Aside from purifying polyhistidine-tagged recombinant proteins, 6xHis tags can also be used for the following applications.
ELISA or Western blot detection. You can use nickel-chelated horseradish peroxidase to facilitate HRP-based detection of His-tagged proteins without antibodies. Alternatively, anti-6xHis antibodies that are highly sensitive and specific for proteins bearing the histidine tag are also available.
Protein interaction pull-down. Nickel agarose resin can be used to purify, identify and measure interactors of His-tagged proteins.
Microplate coating. Nickel- or copper-coated microplates allow fusion proteins to be coated from crude or semi-purified samples for various kinds of plate and reporter assays.
Gel staining. Several immune-analytical methods make use of polyhistidine-tags to detect target proteins through anti-polyhistidine-tag antibodies or by SDS-PAGE.
Fluorescent tagging. Fluorescent hexahistidine tags have also been developed to enable researchers to investigate protein migration and trafficking.