For purification of His-tagged proteins by gravity-flow chromatography

  • One-step purification from crude lysate to >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 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.

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Quantity Product Cat. no. Price Sum
 
Ni-NTA Agarose (25 ml)
25 ml nickel-charged resin (max. pressure: 2.8 psi)
30210
$285.00
$0.00
 
Ni-NTA Agarose (100 ml)
100 ml nickel-charged resin (max. pressure: 2.8 psi)
30230
$970.00
$0.00
 
Ni-NTA Agarose (500 ml)
500 ml nickel-charged resin (max. pressure: 2.8 psi)
30250
$4,174.00
$0.00
The Ni-NTA Agarose is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Protein purification with the Ni-NTA protein purification system.|One-step purification under native conditions.|
|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.)
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Performance

Ni-NTA Agarose provides Ni-NTA coupled to a Sepharose CL-6B support and offers high binding capacity and minimal nonspecific binding (see figure One-step purification under native conditions). 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 His-tagged proteins from E. coli .

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 (consecutive or alternating) — the His 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 His-tagged proteins consists of 4 stages: 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). 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 His/Ni-NTA interaction
DenaturantsDetergents 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
8 M Urea 2% Tween 20 10 mM DTT 20% Ethanol 5 mM CaCl2 or 100 mM NaOH
1% CHAPS 20 mM TCEP 20 mM Imidazole 2 M NaCl

Applications

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

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
Feature
Specifications
Applications Proteomics
Bead size 45–165 µm
Binding capacity Up to 50 mg/ml
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
Tag 6xHis tag
Yield 100 µg – 100 mg

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