For His-tagged protein purification using liquid chromatography systems

  • 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, 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.

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Please note: 30725: Ni-NTA Superflow Cartridges (100 x 1 ml) is no longer available and has been superseded by the 30721: Ni-NTA Superflow Cartridges (5 x 1 ml). For more information, please contact QIAGEN Technical Services.
Quantity Product Cat. no. Price Sum
 
Ni-NTA Superflow Cartridges (5 x 1 ml)
5 cartridges pre-filled with 1 ml Ni-NTA Superflow: for automated purification of His-tagged proteins using liquid chromatography systems
30721
$160.00
$0.00
 
Ni-NTA Superflow Cartridges (100 x 1 ml)
100 cartridges pre-filled with 1 ml Ni-NTA Superflow: for automated purification of His-tagged proteins using liquid chromatography systems
30725
Inquire
 
Ni-NTA Superflow Cartridge (1 x 5 ml)
1 cartridge pre-filled with 5 ml Ni-NTA Superflow: for automated purification of His-tagged proteins using liquid chromatography systems
30760
$139.00
$0.00
 
Ni-NTA Superflow Cartridges (5 x 5 ml)
5 cartridges pre-filled with 5 ml Ni-NTA Superflow: for automated purification of His-tagged proteins using liquid chromatography systems
30761
$555.00
$0.00
 
Ni-NTA Superflow Cartridges (100 x 5 ml)
100 cartridges pre-filled with 5 ml Ni-NTA Superflow: for automated purification of His-tagged proteins using liquid chromatography systems
30765
$8,869.00
$0.00

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


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An independent study shows that Ni-NTA loses less nickel than any other tested resin.|Highly reproducible and reliable purification.|Efficient one-step purification of His-tagged proteins using buffers containing a wide variety of additives.|Ni-NTA outperforms other resins to deliver high yields of high-purity protein.|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.|
An independent study shows that Ni-NTA loses less nickel than any other tested resin. Fifty column volumes of buffer containing various additives was passed through a small column containing 100 μl resin from QIAGEN, Supplier G, S, or I. The flowthrough was pooled and a sample sent for analysis by inductively coupled plasma mass spectrometry (ICP-MS) at Dr.Weßling Laboratories, Bochum, Germany according to DIN EN ISO 17025. Native buffer: 50 mM Na phosphate; 300 mM NaCl; 10 mM imidazole, pH 8.0. Denaturing buffer: 100 mM Na phosphate; 10 mM Tris·Cl; 8 M urea.
|His-tagged pGAPase was purified in 10 sequential purification procedures. Column load was 10 ml aliquots of cleared E. coli cell lysate containing 30 mg spiked protein. Between purification runs the column was cleaned in place using 0.5 M NaOH. Groups of three samples show column load, flow-through, and peak elution fraction. M: markers.
|The indicated protein was purified in buffers containing  [A]  reducing agent (10 mM DTT) and  [B]  detergent (1% n-dodecyl-β-D-maltoside)  [C]  Human GBP1 expressed in S. cerevisiae (data kindly provided by Julia Fres, Center for Molecular Medicine, Cologne University, Germany). M:markers; C: cleared lysate; F: flow-through; W: wash; L: lysate; S: soluble fraction; P: pellet; E: elution fractions.
|Ni-NTA outperforms other resins to deliver high yields of high-purity protein. ERK2 was purified using a Ni-NTA Superflow Cartridge or the indicated resin from another supplier, according to manufacturer's instructions. M: markers; C: cleared lysate; F: flow-through; W: wash; E: elution fractions.
||See table 'Micro to large-scale purification of IL-1β for a biopharmaceutical project.'|
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)

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
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).
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.

Feature
Specifications
Applications Proteomics
Bead size 60-160 µm
Binding capacity Up to 50 mg/ml
FPLC Yes
Gravity flow or spin column FPLC or syringe
N- or C-terminal tag Both
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)