Ni-NTA Superflow Columns

For gravity-flow purification of His-tagged proteins

Features

Up to 75 mg highly pure His-tagged protein per column
Ready-to-use columns for gravity-flow purification
Purification under native or denaturing conditions

Useful Resources

(EN) - Important Note for Ni-NTA Users (EN) - Ni-NTA Superflow BioRobot Handbook Manual purification of 6xHis-tagged proteins from E. coli using Ni-NTA Superflow Columns - (EN)

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Ni-NTA Superflow Columns (12 x 1.5 ml)

Cat. No. / ID: 30622

For 12 6xHis-tagged protein preps: 12 polypropylene columns containing 1.5 ml Ni-NTA Superflow

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

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

Ni-NTA Superflow is available in convenient, pre-packed columns for purification of His-tagged proteins from manually prepared cleared lysates, which are applied to Ni-NTA Superflow Columns on a BioRobot vacuum manifold. His-tagged proteins are strongly and selectively bound to Ni-NTA.

Principle

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Superflow Columns, 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 Ni NTA Superflow 96 Columns automated procedure.

See figures

Ni-NTA Superflow 96 Columns automated procedure.

Procedure

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution (see figure ). 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
Denaturants	Detergents	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 CaCl₂	or 100 mM NaOH
	1% CHAPS	20 mM TCEP	20 mM imidazole*	2 M NaCl

See figures

Protein purification with the Ni-NTA Protein Purification System.

Applications

The QIAexpress Ni-NTA Protein Purification System 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

Protein purification with the Ni-NTA Protein Purification System.

Specifications

Features	Specifications
Applications	Proteomics
Tag	6xHis tag
Number of preps per run	1–24 samples per run
Special feature	Cross-contamination-free
Processing	Automated/manual
Support/matrix	Superflow
Gravity flow or spin column	Gravity flow or automated
Binding capacity	5–20 mg/ml
Bead size	60–160 µm
Scale	Large scale
Start material	Cell lysate
Yield	<30 mg

Resources

Download Safety Data Sheets for QIAGEN product components.

For Automated medium and large-scale purification of 6xHis-tagged proteins

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.

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

Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH.

Müller TA; Fleischmann T; van der Meer JR; Kohler HP;

Appl Environ Microbiol; 2006; 72 (7):4853-61 2006 Jul PMID:16820480

FAQ

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

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

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

The binding capacity of Ni-NTA Agarose is the same regardless of the format used. However, the batch procedure (mixing the Ni-NTA resin with lysate or protein sample prior to loading it onto a column, as opposed to loading the sample onto a column pre-packed with Ni-NTA resin) can provide more efficient binding for dilute proteins, since binding can be carried out for an extended period (approximately 1 hour), and resin amounts can be scaled for variable amounts of lysate/protein sample.

FAQ ID -147

The binding capacity of both resins is the same: up to 50mg/ ml mg 6xHis-tagged protein per ml of resin (2500 nmol @ ~20 kDa). The difference between them is the bead support, which determines pressure resistance and flow rate:

Ni-NTA Agarose:

Sepharose CL-6B (bead size 45–165 µm)
max. volumetric: 0.5–1.0 ml/min
max. pressure: 2.8 psi/(0.2bar)
for use with gravity flow only

Ni-NTA Superflow:

Superflow (bead size 60–160 µm)
max. volumetric: 20 ml/min
max. pressure: 140 psi/(10bar)
for use with gravity flow or FPLC

You can find a detailed comparison table in the Appendix at the back of the QIAexpressionist Handbook under the title 'Ni-NTA Matrices'.

FAQ ID -764

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

Compatibility of reagents with Ni-NTA matrices

Reagent	Effect	Comments
Buffer reagents
Tris, HEPES, MOPS	Buffers with secondary or tertiary amines will reduce nickel ions	Up to 100 mM has been used successfully in some cases Sodium phosphate or phosphate-citrate buffer is recommended
Chelating reagents
EDTA, EGTA	Strip nickel ions from resin	Up to 1 mM has been used successfully in some cases, but care must be taken
Sulfhydril reagents
beta-mercaptoethanol	Prevents disulfide cross-linkages	Up to 20 mM
DTT, DTE	Low concentrations will reduce nickel ions	A maximum of 1 mM may be reduce nickel ions used, but beta-mercaptoethanol is recommended
Detergents
Nonionic detergents (Triton, Tween, NP-40, etc.)	Removes background proteins and nucleic acids	Up to 2% can be used
Cationic detergents		Up to 1% can be used
CHAPS		Up to 1% can be used
Anionic detergents (SDS, sarkosyl)		Not recommended, but up to 0.3% has been used success-fully in some cases
Denaturants	Solubilize proteins
GuHCl		Up to 6 M
Urea		Up to 8 M
Amino acids
Glycine		Not recommended
Glutamine		Not recommended
Arginine		Not recommended
Histidine	Binds to Ni-NTA and competes with histidine residues in the 6xHis tag	Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged protein from the Ni-NTA matrix
Other additives
NaCl	Prevents ionic interactions	Up to 2 M can be used, at least 300 mM should be used
MgCl₂		Up to 4 M
CaCl₂		Up to 5 mM
Glycerol	Prevents hydrophobic interaction between proteins	Up to 50%
Ethanol	Prevents hydrophobic interactions between proteins	Up to 20%
Imidazole	Binds to Ni-NTA and competes with histidine residues in the 6xHis tag	Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged
Sodium bicarbonate		Not recommended
Hemoglobin Ammonium Citrate		Not recommended Not recommended Up to 60mM has been used successfully

FAQ ID -49

Yes, please follow either of the QIAGEN Supplementary Protocols:

FAQ ID -967

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

Ni-NTA Superflow Columns

For gravity-flow purification of His-tagged proteins

Features

Useful Resources

Product Details

Principle

See figures

Procedure

See figures

Applications

Supporting data and figures

Protein purification with the Ni-NTA Protein Purification System.

Specifications

Resources

Publications

A highly specific system for efficient enzymatic removal of tags from recombinant proteins.

Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH.

FAQ

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