His•Bind® Purification Kits for Purification 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•Bind® sequence (6, 8 or 10 consecutive histidine residues) binds to divalent cations (Ni2+) immobilized on IDA-based His•Bind® 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, non-denaturing 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-to-handle agaroses for batch and gravity flow columns, and high flow rate Superflow and Fractogel® resins suitable for production scale purification. Several supports are provided pre-charged with Ni 2+, and either NTA or IDA chemistries are available.

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 β-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. 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 co-purification of non-specifically bound proteins.

His•Bind® Matrix Selection Guide

Product Form Capacity Features Applications
Ni-NTA His•Bind® Resin Ni-charged NTA agarose 5-10 mg/mL Minimal Ni2+ leaching Compatible with 20 mM β-ME Small to medium scale Gravity flow column Recommended for eukaryotic extracts
Ni-NTA His•Bind® Superflow Ni-charged NTA Superflow agarose 5-10 mg/mL Minimal Ni2+ leaching Compatible with 20 mM β-ME High flow rates and pressures Small to production scale FPLC or gravity flow column Recommended for eukaryotic extracts
His•Bind® Resin Uncharged IDA agarose 8 mg/mL Reusable many times Compatible with His•Bind® Buffer Kit Small to medium scale Gravity flow column or batch mode
Note: as with any purification matrix, the cleanest separations are achieved when a His•Bind® resin is used near its binding capacity

His•Bind® Purification Kits containing BugBuster® Reagent are available for convenient preparation of soluble cell extracts.

 

Materials

Product No. Description
70666 Ni-NTA His•Bind® Resin
70691 Ni-NTA His•Bind® Superflow Resin
69670 His•Bind® Resin
69755 His•Bind® Buffer Kit
70239 His•Bind® Purification Kit
70751 BugBuster® Ni-NTA His•Bind® Purification Kit
70793 BugBuster® His•Bind® Purification Kit