Overview
Purification of His•Tag fusion proteins by metal chelation chromatography

The His•Bind® family of products offers a wide selection of supports designed for rapid one-step purification of proteins containing the His•Tag® sequence by immobilized metal affinity chromatography (IMAC). The His•Tag sequence (6, 8 or 10 consecutive histidine residues) binds to divalent cations (Ni²⁺) immobilized on NTA*- and IDA-based His•Bind and His•Mag™ resins. After unbound proteins are washed away, the target protein is recovered by elution with either imidazole or slight reduction in pH. This versatile system enables proteins to be purified under gentle, nondenaturing conditions, or in the presence of either 6 M guanidine or urea.

The various His•Bind supports listed in the table below cover many applications for fusion protein purification. Choices include small scale cellulose-based columns and cartridges for convenient handling of multiple samples, bulk easy-tohandle agaroses for batch and gravity flow columns, His•Mag™ Agarose Beads for rapid purification of multiple samples with minimum handling time, and high flow rate Superflow™ and Fractogel® resins suitable for production scale purification. Several supports are provided precharged with Ni²⁺, and either NTA or IDA chemistries are available.

Custom Configurations
His•Bind Resin and His•Mag Agarose Beads can be custom packaged in larger quantities to suit your needs.

* Manufactured by QIAGEN

NTA and IDA Chemistries

With the His•Tag/His•Bind technology, purification is based on the affinity between the neighboring histidines of the His•Tag sequence and an immobilized metal ion (usually Ni²⁺). The metal is held by chelation with reactive groups covalently attached to a solid support. The most commonly used chelators include nitriloacetic acid (NTA) and iminodiacetic acid (IDA), which have four and three sites available for interaction with metal ions, respectively.
The two chemistries confer different properties to the affinity support and conditions used for binding, washing, and elution of target proteins for both native and denaturing conditions. In practice, the additional chelation site available with NTA minimizes leaching of the metal during the purification and is compatible with up to 20 mM b-Mercaptoethanol for reduction of disulfide bonds. The higher metal leaching rates of IDA-based resins in the presence of other chelating or reducing components can produce poor purification results when these products are present in the buffer. However, IDA supports can be recycled many times with no loss in performance. For both types of support, the conditions can be modified to optimize the purification of individual target proteins expressed in specific systems. Most often, the imidazole concentrations of the wash and elution buffers under native conditions are adjusted to minimize copurification of nonspecifically bound proteins.

His•Bind Columns

Designed for convenience, the single-use His•Bind Columns are pre-packed with 1.25-ml bed volume of Ni²⁺-charged His•Bind Resin. Top and bottom frits ensure even buffer flow and minimal disturbance when loading and running the column. Optimal performance is achieved with bacterial lysates prepared using BugBuster® plus Benzonase® Nuclease.

His•Bind and His•Bind Quick Buffer Kits

The His•Bind Buffer Kit is a set of pre-tested buffers designed for use with IDA-based His•Bind Resin for convenient, rapid one-step purification of proteins by metal chelation chromatography. Solutions are included for Ni²⁺ charging, binding, washing, and elution of up to ten 2.5-ml columns. The His•Bind Quick Buffer Kit contains the same components except that the 8X Charge Buffer is not included (the resin is provided pre-charged with Ni²⁺).

Ni-NTA Buffer Kit

The Ni-NTA Buffer Kit provides a convenient set of buffers optimized for purification of His•Tag fusion proteins under native conditions on Ni-NTA His•Bind Resin. These phosphate-buffered solutions differ from the Tris-based solutions used in the His•Bind Buffer Kit. Carefully prepared 4X concentrates are included for binding, washing, and elution according to recommended protocols.