Ni-NTA Superflow 96 BioRobot Kit

For automated, medium-scale purification of 6xHis-tagged proteins

Features

  • Fully automated purification of up to 10 mg protein per well 
  • High-purity protein free from cross-contamination 
  • Purification under native or denaturing conditions 
Ni-NTA Superflow 96 BioRobot Kit (4)

Cat. No. / ID: 969261

For 4 x 96 6xHis-tagged protein preps: 4 QIAfilter 96 Plates, 4 TurboFilter 96 Plates, 1 x 100 ml Ni-NTA Superflow
€1,546.00
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The Ni-NTA Superflow 96 BioRobot Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The Ni-NTA Superflow 96 BioRobot Kit provides automated purification of up to 600 µg recombinant protein from 96 samples in parallel (standard protocol, 5 ml culture volume per sample). Cleared bacterial cell lysates flow onto a filter plate preloaded with Ni-NTA Superflow by the BioRobot workstation. This unique 96-well metal-chelate affinity-chromatography module strongly and selectively binds His-tagged proteins. Ready-to-run protocols are provided for purification under native or denaturing conditions.

Performance

In response to customers’ needs for larger amounts of protein from automated procedures, QIAGEN has adapted the Ni-NTA Superflow 96 BioRobot protocol to enable processing of larger culture volumes and purification of milligram amounts of His-tagged protein per well. Increasing the amount of Ni-NTA Superflow resin used in the procedure to 200 µl per well and an optimized lysis buffer formulation enables up to 25 ml (purification under native conditions) or 15 ml (purification under denaturing conditions) cultures to be processed (high-yield protocols). Cell cultures are transferred to 24-well blocks for processing. The corresponding increase in biomass can deliver in some cases up to 10 mg of pure His-tagged protein per well (see table  and figure  Milligram amounts of His-tagged proteins per well) allowing multiple assays to be carried out on the same batch of protein and reducing the total number of protein preps required, see also figure  Automated expression clone screening. The single-step purification provides highly pure proteins over a wide range of yields, and purification of large protein complexes is possible.

Yields of His-tagged proteins using the adapted Ni-NTA Superflow 96 BioRobot protocol
His-tagged protein Total yield per well
(µg)*
Protein concentration
(mg/ml)†
Green fluorescent protein 4000 6.0
T7 RNA polymerase 1000 1.4
GroES 300 0.4
Chloramphenicol acetyltransferase 2400 4.4
GroEL 740 1.0
Tumor necrosis factor α 1600 2.5
GroES/GroEL 1200 1.5
Cpn-10 170 0.3
* Yield obtained in two 550 µl elution fractions (average of 6 independent purifications). 80% of the protein elutes in the first 550 µl.
† Protein concentration in the first 550 µl elution fraction.
See figures

Principle

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Superflow 96 BioRobot Kit, 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  Protein purification with the Ni-NTA protein purification system).

See figures

Procedure

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution anfd is the basis of the Ni-NTA Superflow BioRobot Kit (see figure  Ni-NTA Superflow 96 BioRobot Kit). 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.

The Ni-NTA Superflow 96 BioRobot Kit is for use with BioRobot 3000, 8000, or 9600 workstations.  .

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 CaCl2or 100 mM NaOH
 1% CHAPS 20 mM TCEP20 mM imidazole 2 M NaCl  

 

See figures

Applications

The QIAexpress Ni-NTA Protein Purification System provides reliable, one-step purification of proteins suitable for many 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

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsProteomics
yield15 µg – 4 mg
startmaterialCell lysate
tag6xHis tag
beadsize60–160 µm
specialfeatureCross-contamination-free
scaleMedium scale
numberofprepsperrun1–96 samples per run
processingAutomated
supportmatrixSuperflow
bindingcapacity5–20 mg/ml

Resources

Kit Handbooks (2)
For Automated medium and large-scale purification of 6xHis-tagged proteins
Supplementary Protocols (2)
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.
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.

FAQ

What are your recommendations for PCR template preparation for use with the EasyXpress Insect Kit II?

We recommend to use the EasyXpress Linear Template Kit Plus to generate PCR products optimized for use in protein expression with the EasyXpress Insect Kit II.

This kit uses specially designed primers to amplify coding DNA sequence and supplement it with regulatory elements required for optimal transcription and translation in cell-free expression systems. In addition, specially designed 5' untranslated regions (UTRs) on the sense adapter primer sequences reduce the formation of secondary structure in the translation initiation region, one of the commonest causes of low expression rates. A His-or Strep-tag II can be added to either terminus, greatly simplifying protein purification and detection after expression.

FAQ ID -1221
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
Can Ni-NTA resins be used to purify protein with an internal His-tag?
Yes, Ni-NTA Agarose and Superflow will bind a 6xHis-tag whether it is located internally or at the C- or N-teminal end of the protein. Note that the His-tag must be exposed for binding at the surface of the protein to allow for efficient purification under native conditions.
FAQ ID -496
Do you have a protocol for manual purification of 6xHis-tagged proteins expressed in E. coli using Ni-NTA Superflow?