Protino® 96 Ni-NTA

Principle / Procedure

Protino® Ni-NTA products enable fast and convenient purification of recombinant polyhistidine-tagged proteins by immobilized metal ion affinity chromatography (IMAC). Proteins from any expression system can be purified under native or denaturing conditions. Binding of protein is based on the interaction between the polyhistidine tag of the recombinant protein and immobilized Ni2+ ions.

Protino® Ni-NTA Agarose consists of the chelating ligand nitrilotriacetic acid (NTA) immobilized on 6 % cross-linked agarose beads that are suitable for batch binding, gravity-flow, and FPLC™ columns. The resin is precharged with Ni2+ ions and therefore ready to use.

Protino® Ni-NTA Agarose uses NTA, which represents the most commonly used chelating ligand in IMAC. NTA is a tetradentate chelator which occupies four of the six binding sites in the coordination sphere of the Ni2+ ion. The remaining two coordination sites are usually occupied by water molecules and can be exchanged with histidine residues of the recombinant protein (Figure 1). This formation of coordination sites has turned out to be optimal for purification of polyhistidine-tagged proteins: Two available binding sites in the coordination sphere of the Ni2+ ion enable tight but reversible selective protein interactions. Chelation of Ni2+ ions by NTA through four coordination positions minimizes metal leaching during purification and increases specificity for polyhistidine-tagged proteins.

Structure of NTA in complex with Ni2+

Features

High-throughput purification of His-tag proteins

• Efficient protein purification using chelating group NTA (nitrilotriacetic acid)
• Unique Protino® Purification Plate for leak-free incubation during all steps
• Plate outlet and MN Wash Plate prevent cross-contamination
• High well-to-well reproducibility
• High capacity and high affinity
• Purification under native and denaturing conditions
• Processing under vacuum or by centrifugation

* Binding capacity will vary for each polyhistidine-tagged protein and strongly depends on chromatographic conditions