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Ni-NTA Superflow Columns

For gravity-flow purification of His-tagged proteins

  • Up to 75 mg highly pure His-tagged protein per column
  • Ready-to-use columns for gravity-flow purification
  • Purification under native or denaturing conditions

Ni-NTA Superflow is available in convenient, pre-packed columns for purification of His-tagged proteins from manually prepared cleared lysates, which are applied to Ni-NTA Superflow Columns on a BioRobot vacuum manifold. His-tagged proteins are strongly and selectively bound to Ni-NTA.

Cat No./ID: 30622
Ni-NTA Superflow Columns (12 x 1.5 ml)
S Fr.592.00
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For 12 6xHis-tagged protein preps: 12 polypropylene columns containing 1.5 ml Ni-NTA Superflow

Ni-NTA Superflow Columns are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Ni-NTA Superflow 96 Columns automated procedure.
Principle

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Superflow Columns, is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins containing an affinity tag of six or more histidine residues — 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. See figure Ni NTA Superflow 96 Columns automated procedure.

Procedure
The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution (see figure 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 His 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
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
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  
* Higher concentrations of imidazole (100–250 mM) are used to elute His-tagged proteins from Ni-NTA resins.
 

 

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
Features
Specifications
Applications Proteomics
Bead size 60–160 µm
Binding capacity 5–20 mg/ml
Gravity flow or spin column Gravity flow or automated
Number of preps per run 1–24 samples per run
Processing Automated/manual
Scale Large scale
Special feature Cross-contamination-free
Start material Cell lysate
Support/matrix Superflow
Tag 6xHis tag
Yield <30 mg

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Kit Handbooks (2)
Supplementary Protocols (2)
The two protocols given below are for the use of the Ni-NTA Superflow 96 BioRobot® Kit in manual procedures. The kit has been specially designed and optimized for automated 6xHis-tagged protein purification on QIAGEN® BioRobot Systems. For more details of the advantages of BioRobot Systems see the Ni-NTA Superflow 96 BioRobot Kit Handbook supplied with the kit or contact one of the QIAGEN Technical Service Departments or local distributors listed on the last page of the handbook.
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The following protocols have been designed for the use of Ni-NTA Superflow Columns on the QIAvac 6S vacuum manifold or in gravity-flow applications on the QIArack. Up to 15 mg 6xHistagged protein can be purified per column from cleared lysate derived from up to 1 liter of (E. coli) bacterial culture.
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