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NTA Agarose

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

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.

Cat No./ID: 30310
NTA Agarose (25 ml)
$344.00
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25 ml uncharged resin (max. pressure 2.8 psi)
The NTA Agarose is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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.

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.

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  


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
Features
Specifications
Applications Proteomics
Bead size 45–165 µm
Binding capacity Up to 50 mg/ml (up to 2.5 µmol @ ~20 kDa* * Based on immobilized nickel ion chromatography; capacities may vary for other ions.
Form null
FPLC Yes
Gravity flow or spin column Gravity flow
Processing Manual/Automated
Scale Large scale
Special feature Batch and column purification
Start material Cell lysate
Support/matrix Sepharose CL-6B
Yield 100 µg – 100 mg
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