For His-tagged protein purification using liquid chromatography systems
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Cat. No. / ID: 30761
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✓ Knowledgeable and professional Product & Technical Support
✓ Fast and reliable (re)-ordering
Ni-NTA Superflow, the most-cited resin used for purification of His-tagged proteins, is available in pre-filled 1 ml and 5 ml cartridges for automated purification on liquid chromatography systems such as the FPLC, ÄkTA, and BioLogic systems, or manual purification using a syringe.
Ni-NTA gives superior performance in delivering high yields of highly purified protein, in comparison to other resins (see figure Ni-NTA outperforms other resins to deliver high yields of high-purity proteins). An independent comparison with other commercially available nickel resins demonstrated that Ni-NTA Superflow shows the lowest level of nickel leaching (see figure An independent study shows that Ni-NTA loses less nickel than any other tested resin). The significance here is that if nickel is leached from the resin, the remaining charged ligands act as an ion exchanger and bind non-tagged proteins that will contaminate elution fractions. The higher stability of Ni-NTA means that even in the presence of 10 mM DTT it can be used to obtain fully active, high-purity proteins (see figure The extra coordination site in Ni-NTA binds nickel ion more tightly than IDA).
As shown in the table, purities are consistently high over all purification scales. Ni-NTA Superflow Cartridges form part of the comprehensive and complementary solutions for His-tagged protein offered by QIAGEN (see figure Scalable purification of nanogram to gram amounts of His-tagged proteins).
| Matrix | Matrix volume |
Culture volume |
Yield | Recovery (%) | Purity* |
|---|---|---|---|---|---|
| Ni-NTA Magnetic Agarose Beads (micro-scale) | 100 μl | 1 ml | 33 μg | ~90% | ~97% |
| Ni-NTA Superflow (small-scale) | 500 μl | 320 ml | 6 mg | ~80% | ~96% |
| Ni-NTA Superflow (medium-scale) | 10 ml | 1.7 l | 109 mg | ~80% | ~98% |
| Ni-NTA Superflow (large-scale) | 100 ml | 18 l | 2 g | >88% | ~97% |
An independent study shows that Ni-NTA loses less nickel than any other tested resin.
The extra coordination site (arrowed) in Ni-NTA binds nickel more tightly than IDA (the ligand used in many competitor resins).
Scalable purification of nanogram to gram amounts of His-tagged proteins.
Ni-NTA matrices are the affinity chromatography solution of choice for purifying His-tagged proteins (see figure Efficient one-step purification of His-tagged proteins). Their high stability means that they are compatible with a wide range of buffer components, including strong denaturants, detergents, and even reducing agents (see table Reagents compatible with the His/Ni-NTA interaction). This flexibility enables researchers to develop optimal purification schemes while still benefiting from the excellent separation characteristics delivered by Ni-NTA, often making a second chromatographic step unnecessary.
| Denaturants | Detergents | Reducing agents | Others | Salts | 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 | 20 mM TCEP | 20 mM imidazole | 2 M NaCl |
Ni-NTA matrices can be used to scale up purification of His-tagged proteins for structural studies (e.g., using protein crystallography or NMR), or to produce gram amounts for biopharmaceutical production.
| Features | Specifications |
| Applications | Proteomics |
| Gravity flow or spin column | FPLC or syringe |
| Binding capacity | Up to 50 mg/ml |
| FPLC | Yes |
| N- or C-terminal tag | Both |
| Bead size | 60-160 µm |
| Processing | Manual/Automated |
| Scale | Large scale |
| Start material | Cell lysate |
| Support/matrix | Superflow |
| Tag | 6xHis tag |
| Yield | Up to 50 mg (1 ml cartridge), up to 250 mg (5 ml cartridge) |
