Ni-NTA Magnetic Agarose Beads

For high-throughput, micro-scale purification of His-tagged proteins and versatile magnetocapture assays using His tags

Features

  • Directed presentation for enhanced signal-to-noise and reproducibility
  • Wide range of binding capacities by varying the number of beads
  • Effective screening procedures even with crude cell lysates
  • Ideal for study of biomolecular interactions
  • Options for full automation
Ni-NTA Magnetic Agarose Beads (6 x 1 ml)

Cat. No. / ID: 36113

6 x 1 ml nickel-charged magnetic agarose beads (5% suspension)
Ni-NTA Magnetic Agarose Beads are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Ni-NTA Magnetic Agarose Beads are magnetic particles coated with Ni-NTA Agarose affinity purification matrix. They are used for immobilizing and 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. Once proteins are bound, the beads can be precipitated using a magnet, washed, and proteins eluted in small volumes of buffer under native or denaturing conditions.

Principle

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Magnetic Agarose Beads (see figure  Micrograph of Ni-NTA Magnetic Agarose Beads) 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 (see figure  Protein purification with the Ni-NTA protein purification system). 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 figures

Procedure

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution (see figure  Micro-scale protein purification). 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
DenaturantsDetergentsReducing 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 TCEP20 mM imidazole 2 M NaCl  

 

 

See figures

Applications

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Magnetic Agarose Beads, 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

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsProteomics
processingAutomated/manual
yield<10 mg protein per column
beadsize20–70 µm
supportmatrixMagnetic agarose beads
tag6xHis tag
specialfeatureHigh throughput (96-well)
startmaterialCell lysate
bindingcapacityUp to 2 mg/ml suspension (5%)
scaleMicro scale

Publications

A highly specific system for efficient enzymatic removal of tags from recombinant proteins.
Schäfer F; Schäfer A; Steinert K;
J Biomol Tech; 2002; 13 (3):158-71 2002 Sep PMID:19498979
Modulating RssB activity: IraP, a novel regulator of sigma(S) stability in Escherichia coli.
Bougdour A; Wickner S; Gottesman S;
Genes Dev; 2006; 20 (7):884-97 2006 Apr 1 PMID:16600914
Bending fatigue study of nickel-titanium Gates Glidden drills.
Luebke NH; Brantley WA; Alapati SB; Mitchell JC; Lausten LL; Daehn GS;
J Endod; 2005; 31 (7):523-5 2005 Jul PMID:15980713
Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay.
Day-Storms JJ; Niranjanakumari S; Fierke CA;
RNA; 2004; 10 (10):1595-608 2004 Aug 30 PMID:15337847
Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.
Suer S; Sickmann A; Meyer HE; Herberg FW; Heilmeyer LM Jr;
Eur J Biochem; 2001; 268 (7):2099-106 2001 Apr PMID:11277933

FAQ

How can I purify very small amounts of 6xHis-tagged protein using Ni-NTA technology?

When working with small amounts of 6xHis-tagged protein in dilute solution, such as proteins expressed in mammalian cells or secreted into cell-culture medium, we recommend using Ni-NTA Magnetic Agarose Beads.

The total binding capacity of Ni-NTA Magnetic Agarose Beads is 3 µg of protein per 10 ul of magnetic bead suspension. Adjusting the amount of beads to the amount of 6xHis-tagged protein to be captured is crucial for optimal performance. The small elution volumes used provide high 6xHis-tagged protein concentrations, and allow detection of the purified proteins using Coomassie-stained SDS polyacrylamide gels.

Please see protocol 15 in the QIAexpressionist Handbook for detailed descriptions of a procedure to purify 6xHis-tagged proteins from transfected mammalian cells.

FAQ ID -134
How can I remove imidazole from a protein sample?
Imidazole does not interfere with most downstream applications and therefore does not need to be removed. If it is necessary to remove the imidazole (e.g., for some sensitive enzyme assays), it can be easily achieved by dialysis, precipitation (e.g., ammonium sulfate), or ultrafiltration.
FAQ ID -91
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 the purification of 6xHis-tagged proteins using BioSprint?
Yes, please follow the Supplementary Protocol 'Purification of 6xHis-tagged proteins using the BioSprint 96 Workstation' (BS17).
FAQ ID -1167
How can I eliminate contaminating protein in my Ni-NTA 6xHis-tag protein purification?
  • Use 10-20 mM imidazole in the lysis and wash buffers (both for native and denaturing conditions). Optimal imidazole concentrations have to be determined empirically.
  • Increase the NaCl concentration (up to 2 M) in the purification buffers to reduce the binding of contaminants as a result of nonspecific ionic interactions.
  • Add ß-mercaptoethanol (up to 20 mM) to the lysis buffer to prevent copurification of host proteins that may have formed disulfide bonds with the protein of interest during cell lysis.
  • Add detergents such as Triton X-100 and Tween 20 (up to 2%), or additives such as glycerol (up to 50%) or ethanol (up to 20%) to reduce nonspecific binding to the matrix due to nonspecific hydrophobic interactions.
  • Reduce the amount of Ni-NTA matrix. Low-affinity binding of background proteins will be reduced by matching the total binding capacity of Ni-NTA matrix with the expected amount of 6xHis-tagged protein.
FAQ ID -102