Ni-NTA Agarose

For purification of 6xHis-tagged proteins by gravity-flow chromatography

• One-step purification from crude lysate to greater than 95% pure protein
• High binding affinity and high capacity
• Choice of purification under native or denaturing conditions
• Precharged, ready-to-use matrices for any scale of purification
• Automated purification and assay protocols

Ni-NTA Agarose provides Ni-NTA coupled to a Sepharose CL-6B support and offers high binding capacity and minimal nonspecific binding. This material has excellent handling properties for most scales of batch and column purification. Ni-NTA Agarose is available separately or as a component of QIAexpress Kits, which are complete kits for efficient expression and purification of 6xHis-tagged proteins from E. coli .

One-Step Purification under Native Conditions

Human serum response factor (SRF) was expressed in HeLa cells carrying a vaccinia virus vector and purified using Ni-NTA Agarose with different imidazole concentrations in the wash and elution steps. Proteins were visualized by silver staining. CL: cell lysate; FT: flow-through; W1: 0.8 mM wash; W2 & W3: 8 mM wash; W4 & W5: 40 mM wash; E1 & E2: 80 mM elution. (Data kindly provided by H. Stunnenberg, EMBL, Heidelberg, Germany.)

Principle

The QIAexpress Ni-NTA Protein Purification System is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins containing an affinity tag of six consecutive histidine residues — the 6xHis tag. This 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. The QIAexpress system can be used to purify His-tagged proteins from any expression system including baculovirus, mammalian cells, yeast, and bacteria.

Procedure

The purification of 6xHis-tagged proteins consists of 4 steps: cell lysis, binding, washing, and elution . Purification of recombinant proteins using the QIAexpress system 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 . Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH.

Reagents Compatible with the 6xHis/Ni-NTA Interaction

Applications

The QIAexpress Ni-NTA Protein Purification System provides reliable, one-step purification of proteins suitable for any application, including:

• Structural and functional investigations
• Crystallization for determination of three-dimensional structure
• Assays involving protein–protein and protein–DNA interactions
• Immunization to produce antibodies

Ni-NTA matrices can also be used to bind 6xHis-tagged proteins as immobilized affinity ligands to:

• Study molecular interactions with nucleic acids and binding proteins
• Purify antibodies
• Isolate nontagged, interacting subunits or nucleic acids
• Investigate ligand–receptor interactions