The Histidine Tag
The DNA sequence specifying a string of six to nine histidine residues is frequently used in vectors for production of recombinant proteins. The result is expression of a recombinant protein with a 6xHis or poly-His tag fused to its N- or C-terminus.
Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially available for use in assay methods involving His-tagged proteins. In either case, the tag provides a means of specifically purifying or detecting the recombinant protein without a protein-specific antibody or probe.
Overview of Affinity Purification
Overview of Protein Expression
Overview of Pull-down Assays
Immobilized Metal Affinity Chromatography (IMAC)
For affinity purification, beaded agarose supports can be derivatized with chelating groups to immobilize the desired metal ions. This is know 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. The resulting resin is typically called (using nickel as the example metal) Ni-chelate agarose or Ni-IDA agarose. Packed into suitable columns or chromatography cartridges, such resins provide for purification of 1 to 10 milligrams of His-tagged protein per millilter 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. However, reducing agents, oxidizing agents and chelators (e.g., EDTA) are not compatible with this 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.
Protein Purification Handbook
IMAC resins and kits for
His-tagged fusion protein
Nickel, Cobalt and Copper
Nickel is the most widely available metal ion for purifying histidine-tagged proteins. Nickel generally provides good binding efficiency to His-tagged proteins but also tends to bind nonspecifically to endogenous proteins that contain histidine clusters. As stated above, a small amount of imidazole in the binding/wash buffer helps to control off-target binding.
Cobalt exhibits a more specific interaction with histidine tags, resulting in less nonspecific interaction. For this reason, cobalt is the preferred divalent cation for purifying His-tagged proteins when high purity is a primary concern.
Copper ions bind His tags more strongly than cobalt or nickel. This provides the highest possible binding capacity but also the poorest specificity. For this reason, copper IMAC is usually used only for binding applications in which purification is not the objective (e.g., plate-coating of an already-purified His-tagged protein for use in an assay). See below.
HisPur Cobalt Resin, Columns, Cartridges and Purification Kits
HisPur Cobalt Superflow Agarose
HisPur Ni-NTA Resin, Columns, Cartridges and Purification Kits
HisPur Ni-NTA Superflow Agarose
All IMAC resins and kits
Other His-tagged Fusion Protein Techniques
Besides affinity purification, other applications for His-tagged fusion proteins are made possible with the aid of IMAC-type chemistries or His-tag-specific antibodies:
- Microplate coating: nickel- or copper-coated microplates enable fusion proteins to be coated from crude or semi-purified samples for plate and reporter assays of various kinds.
- ELISA or Western blot detection: nickel-chelated horseradish peroxidase (Thermo Scientifiic HisProbe HRP) enables HRP-based detection of His-tagged proteins without antibodies. Alternatively, anti-6xHis antibodies are also available.
- Gel staining: a metal-based fluorescent stain enables detection of His-tagged proteins in SDS-PAGE.
- Protein interaction pull-down: nickel agarose resin can be used to purify, identify and measure interactors of His-tagged proteins.
HisProbe HRP Reagent and Kit
6xHis Protein Tag Staining Kit
His-Tag Pull-Down Kit
Search for Anti-Tag Antibodies
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