Ni-NTA Agarose

For purification of His-tagged proteins by gravity-flow chromatography

Features

  • One-step purification from crude lysate to >95% pure protein
  • High binding affinity and high capacity
  • Choice of purification under native or denaturing conditions
  • Precharged, ready-to-use matrices for any scale of purification
  • Automated purification and assay protocols
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Ni-NTA Agarose (25 ml)

Cat. No. / ID: 30210

25 ml nickel-charged resin (max. pressure: 2.8 psi)
Quantity
25 ml
100 ml
500 ml
The Ni-NTA Agarose is intended for molecular biology applications. This product is 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 Agarose is an affinity chromatography matrix for 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. Cleared cell lysates are loaded onto the matrices. His-tagged proteins are bound, and other proteins pass through the matrix. After washing, His-tagged proteins are eluted in buffer under native or denaturing conditions.

Performance

Ni-NTA Agarose provides Ni-NTA coupled to a Sepharose CL-6B support and offers high binding capacity and minimal nonspecific binding (see figure  One-step purification under native conditions). This material has excellent handling properties for most scales of batch and column purification. Ni-NTA Agarose is available separately or as a component of QIAexpress Kits, which are complete kits for efficient expression and purification of His-tagged proteins from E. coli .

See figures

Principle

The QIAexpress Ni-NTA Protein Purification System is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins which contain an affinity tag of six or more histidine residues (consecutive or alternating) — 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.

Procedure

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution (see  Protein purification with the Ni-NTA protein purification system). Purification of recombinant proteins using the QIAexpress system does not depend on the 3-dimensional structure of the protein or 6xHis 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
DenaturantsDetergents 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

Applications

Ni-NTA matrices provide reliable, one-step purification of His-tagged proteins suitable for any application, including:

  • Structural and functional investigations
  • Crystallization for determination of three-dimensional structure
  • Protein–protein and protein–DNA interaction assays
  • Immunization to produce antibodies
  • Scaling up purification to production scale

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsProteomics
Tag6xHis tag
Bead size45–165 µm
Special featureBatch and column purification
FPLCNo
ProcessingManual/Automated
Support/matrixSepharose CL-6B
Start materialCell lysate
ScaleLarge scale
Binding capacityUp to 50 mg/ml
Gravity flow or spin columnGravity flow
Yield100 µg – 100 mg

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
Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.
Block H; Kubicek J; Labahn J; Roth U; Schäfer F;
Protein Expr Purif; 2007; 57 (2):244-54 2007 Oct 17 PMID:18053740
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
Evidence for two modes of development of acquired tumor necrosis factor-related apoptosis-inducing ligand resistance. Involvement of Bcl-xL.
Song JJ; An JY; Kwon YT; Lee YJ;
J Biol Chem; 2006; 282 (1):319-28 2006 Nov 15 PMID:17110373

FAQ

What are your recommendations for PCR template preparation for use with the EasyXpress Insect Kit II?

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
Are the buffers in the Ni-NTA Fast Start Kit the same as the ones for use with Ni-NTA purchased separately?

The buffers of the Ni-NTA Fast Start Kit are based on recipes for the respective buffers for purification of 6xHis-tagged proteins under native or denaturing conditions listed in the QIAexpressionist handbook. Specific components have been added for optimized performance. The exact composition of the buffers in the Ni-NTA Fast Start Kit is confidential. However, the buffers listed in the Appendix Section of the QIAexpressionist are compatible with the Ni-NTA Fast Start Kit, and can also be used.

FAQ ID -791
Is it possible to isolate both RNA and recombinant 6xHis-tagged protein from the same sample?
We have no experimental data for this application. However, buffer RLT of the RNeasy Kits for RNA isolation does not contain substances incompatible with Ni-NTA purification of His-tagged proteins. It should be possible to first extract RNA from a sample by following the RNeasy procedure, save the flow-through from the binding step as well as from the RW1 wash, and apply the combined fractions onto a Ni-NTA column for binding of His-tagged proteins. Follow our recommendations for purification of 6xHis-tagged proteins using Ni-NTA resins outlined in the QIAexpressionist handbook.
FAQ ID -532
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
Should I use Ni-NTA Agarose in column or batch format for purification of 6xHis-tagged proteins?
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
What is the difference between Ni-NTA Agarose and Ni-NTA Superflow?

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
Can I reuse the Ni-NTA Agarose and Ni-NTA Superflow resins?

The reuse of Ni-NTA Agarose and Ni-NTA Superflow resins depends on the nature of the sample and should only be performed with identical recombinant proteins. We recommend a maximum of 5 runs per column. After use the resin should be washed for 30 minutes with 0.5 M NaOH. Ni-NTA matrices should be stored in 30% ethanol to inhibit microbial growth.

If the Ni-NTA matrix changes from light blue to brownish-gray, the regeneration procedure described in the Appendix of the QIAexpressionist Handbook in section 'Reuse of Ni-NTA Resin' is recommended.

FAQ ID -802
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
What are the compatibilities of different reagents with Ni-NTA matrices?

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
How can I check if any residual proteins remain on the Ni-NTA Agarose matrix after elution?
Ni-NTA Agarose may be boiled in SDS-PAGE sample buffer to release any protein that remains on the matrix following elution. All proteins, regardless of whether they bind to Ni-NTA or to the agarose-moiety, will be recovered by this procedure.
FAQ ID -324
3351 - What is the upper limit for protein size that can bind to Ni-NTA agarose resin?
Ni-NTA Agarose has an exclusion size of approximately 4 x 10e7 Da. This is sufficient to allow even extremely large proteins to enter the cavities in the bead surface for binding to Ni-NTA residues.
FAQ ID - 3351
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
3352 - What are the size ranges of Ni-NTA particles?
Ni-NTA Agarose beads are approximately 45-165 µm, Ni-NTA Superflow beads range from 60-160 µm and  Ni-NTA Magnetic Agarose Beads are between 20-70 µm in diameter.
FAQ ID - 3352
Why do you recommend using Triton X for the purification of 6xHis-tagged protein?

Nonionic detergents such as Triton X-100 (0.1 - 1%) and Tween 20 (up to 2%) can be used to reduce non-specific binding of contaminating proteins due to non-specific hydrophobic or ionic interactions. They will have no effect on the binding of 6xHis-tagged protein to the Ni-NTA resin when used within the recommended concentration range.

Optimal concentrations for these additives to binding and wash buffers should be determined empirically for each purification protocol and protein.

-100
How can I be sure that I am harvesting my induced bacterial culture at the best time point for protein expression?

To optimize the expression of a given recombinant protein, a time-course analysis of the level of protein expression in the induced culture is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the 6xHis-tagged protein present at various times after induction in the soluble and insoluble fractions, the optimal induction period can be established, and the bacterial culture can be harvested at this time. It may be useful to perform plasmid Mini preparations on culture samples during the time-course to enable monitoring of plasmid (expression construct) maintenance.

Below, you can see an example of a time course of recombinant protein expression using the QIAexpress System. You can find this information also in the Section 'Expression in E. coli' in the QIAexpressionist Handbook. The handbook is an important resource for useful background information and protocols. For instructions on how to isolate protein from the soluble and insoluble fractions of induced cultures please see Protocol 14. "Protein minipreps of 6x His-tagged proteins from E. coli under native conditions" and Protocol 19. "6xHis-tagged protein minipreps under denaturing conditions."

 

 

 

Time course of expression using the QIAexpress System. Expression of 6xHis-tagged DHFR was induced with 1 mM IPTG. Aliquots were removed at the times indicated and purified on Ni-NTA Agarose under denaturing conditions. Proteins were visualized by Coomassie staining. Yields per liter culture were 2.8, 5.5,12.3, 33.8, and 53.9 mg, respectively. ■A Crude cell lysate; ■B purification with Ni-NTA. 1: flow-through, 2 & 3: first and second eluates; M: markers; C: noninduced control.

 

 

FAQ ID -788