Ni-NTA Superflow Cartridges

For His-tagged protein purification using liquid chromatography systems

Features

  • Most robust one-step purification over the widest range of conditions
  • High yields of high-purity protein, with up to 50 mg/ml
  • Fast, easy, and reproducible processing on any LC system
Ni-NTA Superflow Cartridges (5 x 5 ml)

Cat. No. / ID: 30761

5 cartridges pre-filled with 5 ml Ni-NTA Superflow: for automated purification of His-tagged proteins using liquid chromatography systems
Ni-NTA Superflow Cartridges are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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.

Performance

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).

Micro- to large-scale purification of IL-1β for a biopharmaceutical project
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%
* Determined using Agilent Bioanalyzer (Protein 50 LabChip Kit)

See figures

Principle

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.

Reagents compatible with the His/Ni-NTA interaction.
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
See figures

Procedure

The robust Superflow matrix allows flow rates up to 10 ml/min (1 ml cartridges) and 40 ml/min (5 ml cartridges), speeding the purification procedure and increasing throughput. The cartridges are quickly and easily connected to liquid chromatography systems or a syringe for manual purification. The purification process is highly reproducible, guaranteeing the same high quality protein preparations time after time (see figure  Highly reproducible and reliable purification).
See figures

Applications

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.

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsProteomics
gravityfloworspincolumnFPLC or syringe
bindingcapacityUp to 50 mg/ml
fplcYes
norcterminaltagBoth
beadsize60-160 µm
processingManual/Automated
scaleLarge scale
startmaterialCell lysate
supportmatrixSuperflow
tag6xHis tag
yieldUp to 50 mg (1 ml cartridge), up to 250 mg (5 ml cartridge)

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
Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor.
Yeliseev A; Zoubak L; Gawrisch K;
Protein Expr Purif; 2006; 53 (1):153-63 2006 Dec 12 PMID:17223358

FAQ

Can the Ni-NTA Superflow Cartridges and/or Strep-Tactin Cartridges be connected in series?

Yes, Ni-NTA and Strep-Tactin Superflow Cartridges can be connected in series.

The Union M6 female/1/16’’ male connector from GE (Code No. 18-3858-01) can be used for this, for example.

 

FAQ ID -1606
How fast is the 6xHis-tagged protein purification process using Ni-NTA Superflow Cartridges?

A maximum of 1 hour is needed for the complete purification process with Ni-NTA Superflow Cartridges when recommended flow rates are applied. This is true for 1 ml and 5 ml cartridges.

Details for a typical 1 ml column run:

  • Equilibration (5 column volumes (cv)): 5 min
  • Load (10 ml Cleared Lysate): 10 min
  • Wash (10 cv): 10 min
  • Elution (10 cv): 10 min
  • optional: Cleaning-in-place (0.5 M NaOH, 0.5 ml/min, 7.5 cv): 15 min  
FAQ ID -1605
What is the binding capacity of the Ni-NTA Superflow Cartridges?

The binding capacity of Ni-NTA resins is protein dependent. We guarantee a binding capacity of up to 20 mg/ml of Ni-NTA Superflow for every 6xHis-tagged protein (up to 20 mg per 1 ml Ni-NTA Superflow Cartridge).

However, we have examples where the binding capacity is higher (e.g.: 6xHis-CAT: 30 mg/ml resin; 6xHis-GFP: 55 mg/ml resin).

  • Binding capacity for the 1 ml Ni-NTA Superflow Cartridge: 20 mg
  • Binding capacity for the 5 ml Ni-NTA Superflow Cartridge: 100 mg

 

FAQ ID -1603
Can the Ni-NTA and Strep-Tactin Superflow Cartridges be reused?

If the same protein is purified, the Ni-NTA and Strep-Tactin Superflow Cartridges can be used more than once. Cleaning in place (CIP) is recommended between purifications (0.5 M NaOH, 0.5 ml/min, 7.5 cv for 15 min).

However, for sequential purification of different proteins we recommend to use different cartridges. Please note that cartridges cannot be opened!

 

FAQ ID -1607