NTA Agarose

For efficient immobilized-metal affinity chromatography (IMAC) using gravity-flow chromatography

Features

  • High binding affinity and high capacity
  • Choice of purification under native or denaturing conditions
  • Fine-tuning of purification strategies through flexible choice of metal ion

Product Details


Uncharged NTA Agarose allows researchers to choose the metal ion they use for IMAC procedures, allowing fine-tuning of purification strategies. Metal ions such as Ni 2+, Cu 2+, Zn2+, or Co2+ are efficiently immobilized for IMAC purification of metal-binding proteins. NTA has four chelating sites to bind metal ions more tightly than other metal-chelating purification systems. This reduces metal-ion leaching and nonspecific binding and leads to increased protein binding capacity and purer preparations. A proprietary spacer that links NTA to the sepharose support provides optimal accessibility to metal ions, increasing protein-binding capacity.

Performance

NTA Agarose enables efficient immobilized-metal affinity chromatography (IMAC) using gravity-flow chromatography. NTA has four chelation sites for nickel ions, which results in tighter binding of nickel compared with metal-chelating purification systems that only have three sites available for interaction with metal ions. The extra chelation site prevents nickel-ion leaching, assuring high binding affinity, resulting in a greater binding capacity and protein preparations with greater purity (see figure " One-step purification under native conditions") than those obtained using other metal-chelating purification systems.

See figures

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 (see figure " One-step purification under native conditions") 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.

See figures

Procedure

The purification of 6xHis-tagged proteins consists of 4 steps: cell lysis, binding, washing, and elution (see " Protein purification with the Ni-NTA protein purification system"). 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 (see table "Reagents compatible with the 6xHis/Ni-NTA interaction"). 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
 Denaturants  Detergents  Reducing agents  Others Salts For long-term storage
 6 M Gu·HCl  2% Triton X-100  20 mM β-ME  50% glycerol 4 M MgCl2 Up to 30% ethanol or 100 mM NaOH
 8 M Urea  2% Tween 20  10 mM DTT  20% ethanol 5 mM CaCl2
   1% CHAPS    20 mM imidazole 2 M NaCl  


See figures

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 (see QIAexpress Assay System)
  • 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

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsProteomics
bindingcapacityUp to 50 mg/ml (up to 2.5 µmol @ ~20 kDa* * Based on immobilized nickel ion chromatography; capacities may vary for other ions.
beadsize45–165 µm
scaleLarge scale
fplcYes
gravityfloworspincolumnGravity flow
specialfeatureBatch and column purification
supportmatrixSepharose CL-6B
form50% suspension in 30% ethanol
processingManual/Automated
yield100 µg – 100 mg
startmaterialCell lysate

Resources

FAQ

How do I prevent bubbles from forming in my Ni-NTA agarose column?
Gas bubbles may form when the resin undergoes a temperature change. To alleviate this problem, degas the Ni-NTA agarose in equilibration buffer or simply keep the agarose at a constant temperature to keep all the gases in solution.
FAQ ID -285
What are the features and benefits of the QIAexpress 6xHis Tag System?

FEATURES BENEFITS
The interaction of the 6xHis tag with Ni-NTA matrices is conformation independent One-step purification can be carried out under native or denaturing conditions
Mild elution conditions can be used Binding, washing, and elution are highly reproducible, and have no effect on protein structure. Pure protein products are ready for direct use in downstream applications
The 6xHis tag is much smaller than other commonly used tags 6xHis tags can be used in any expression system. The Tag does not interfere with the structure and function of the recombinant protein
The 6xHis tag is uncharged at physiological pH The 6xHis tag does not interfere with secretion
The 6xHis tag is poorly immunogenic The recombinant protein can be used without prior removal of the tag as an antigen to generate antibodies against the protein of interest
Using Factor Xa Protease, 6xHis tag can be easily and efficiently removed The detagged protein can be used for crystallographical or NMR studies where removal of the 6xHis tag may be preferred
Some QIAexpress vectors feature a 6xHis-dihydrofolate reductase tag (6xHis-DHFR tag) Small peptides fused to the 6xHis DHFR tag are stabilized while being expressed. The 6xHis-DHFR tag is not highly immunogenic in mouse and rat, so that peptides fused to the tag can be used directly for immunizations or epitope mapping

 

FAQ ID -193