Qiagen, 30410, Ni-NTA Superflow (25 ml)

25 mL nickel-charged resin (max. pressure: 140 psi)

Features

- Superior mechanical stability, outstanding flow characteristics and high dynamic binding capacity
- Convenient, one-step purification of 6x His-tagged proteins
- Allows high flow rates and pressures for efficient production-scale and FPLC applications

Product Details

Ni-NTA Superflow is an affinity chromatography matrix for purifying recombinant proteins carrying a His tag.

Histidine residues in the His tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Cleared cell lysates are loaded onto the matrices. His-tagged proteins are bound, and other proteins pass through the matrix. After washing, His-tagged proteins are eluted in buffer under native or denaturing conditions.

Performance

Ni-NTA Superflow is comprised of Ni-NTA coupled to Superflow resin. It combines superior mechanical stability with outstanding flow characteristics and high dynamic binding capacity. The capacity for 6xHis-tagged proteins is 5–10 mg/mL. This resin allows onestep purification of 6xHis-tagged proteins using high flow rates and pressures for efficient production-scale and FPLC applications.

Principle

The QIAexpress Ni-NTA Protein Purification System is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins which contain an affinity tag of six or more histidine residues — the His tag. The technology allows one-step purification of almost any His-tagged protein from any expression system under native or denaturing conditions.

NTA, which has four chelation sites for nickel ions, binds nickel more tightly than metal-chelating purification systems that only have three sites available for interaction with metal ions. The extra chelation site prevents nickel-ion leaching and results in a greater binding capacity and protein preparations with higher purity than those obtained using other metal-chelating purification systems. Ni-NTA Superflow can be used to purify His-tagged proteins from any expression system, including baculovirus, mammalian cells, yeast, and bacteria.

Procedure

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution. Purification of recombinant proteins using Ni-NTA Superflow does not depend on the 3-dimensional structure of the protein or 6xHis tag. This allows one-step protein purification under either native or denaturing conditions, from dilute solutions and crude lysates.

Strong denaturants and detergents can be used for efficient solubilization and purification of receptors, membrane proteins, and proteins that form inclusion bodies. Reagents that allow efficient removal of nonspecifically binding contaminants can be included in wash buffers (see table).

Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH.

Applications

- Structural and functional investigations
- Crystallization for determination of three-dimensional structure
- Protein-protein and protein-DNA interaction assays
- Immunization to produce antibodies
- Scaling up purification to production scale

Ni-NTA matrices provide reliable, one-step purification of His-tagged proteins suitable for any application, including:

For Ni-NTA Superflow, a Drug Master File (DMF) containing product characteristics, chromatographic parameters, chemical and physical information, Quality Control specifications, safety and toxicity information, and standard purification protocols is available for support with regulatory issues.