Ni-NTA Spin System

For fast, small-scale purification of His-tagged proteins

Features

  • Up to 300 μg His-tagged protein per column in as little as 15 minutes
  • Purification under native and denaturing conditions
  • Up to 95% homogeneity in one step
  • Ready-to-use spin columns for rapid automated or manual processing
Ni-NTA Spin Kit (50)

Cat. No. / ID: 31314

50 Ni-NTA Spin Columns, Reagents, Buffers, Collection Tubes, 1 μg Control Expression Plasmid
KitColumn
Ni-NTA Spin Kit
Ni-NTA Spin Column
Ni-NTA Spin System are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Ni-NTA silica combines Ni-NTA with a macroporous silica support material optimized to suppress nonspecific hydrophobic interactions. Ni-NTA Spin Columns (His-protein purification spin columns) in the Ni-NTA Spin Kit and available separately provide Ni-NTA silica in a convenient microspin format for easy preparation of multiple samples in parallel. They provide a simple method for functional screening of engineered proteins, selection of clones expressing full-length translation products and comparison of expression levels. Each spin column can purify up to 300 µg of His-tagged protein. Like all Ni-NTA matrices, Ni-NTA spin columns can be used for one-step protein purification under native or denaturing conditions. The Ni-NTA Spin Kit is a complete kit for spin purification of His-tagged proteins. It can be automated on the QIAcube Connect.

Performance

Ni-NTA Spin Columns (His-protein purification spin columns), also included in the Ni-NTA Spin Kit, allow reproducible fast automated purification (see figure  “Reproducible automated purification”) at different expression levels (see figure  “Purification at different expression levels”).

See figures

Principle

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Spin Columns and Ni-NTA Spin Kit, 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.

Procedure

The purification of His-tagged proteins consists of 4 steps: cell lysis, binding, washing and elution (see figure  “Ni-NTA Spin Column 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 His tag. This allows one-step protein purification under either native or denaturing conditions, from dilute solutions and crude lysates. Up to 600 μl of a cell lysate are loaded onto a Ni-NTA spin column. A quick 2-minute spin binds the tagged protein to Ni-NTA silica, while most of the untagged proteins flow through. After a wash step, purified protein is eluted under mild conditions (such as pH reduction to 5.9, or addition of 100-500 mM imidazole) in a volume of 100-300 μl. Removal of the His tag is usually unnecessary since it is small and rarely immunogenic. The purified protein is ready for immediate use. Proteins can be purified from multiple small-scale expression cultures in around 30 minutes (manual procedure) or approximately 60 minutes (automated QIAcube Connect procedure). 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 Ni-NTA–His interaction

Reagent
6 M guanidine HCl
8 M urea
2% Triton X-100
2% Tween 20
1% CHAPS
20 mM β-ME
10 mM DTT
50% glycerol
20% ethanol
2 M NaCl
4 M MgCl2
5 mM CaCl2
≤20 mM imidazole
20 mM TCEP
See figures

Applications

The QIAexpress Ni-NTA Protein Purification System, including Ni-NTA Spin Columns and the Ni-NTA Spin Kit, 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 interaction
  • Immunization to produce antibodies

Comparison of Ni-NTA Spin Columns and the Ni-NTA Spin Kit

Features Ni-NTA Spin Columns Ni-NTA Spin Kit
Applications Proteomics Proteomics
Bead size 16–24 µm 16–24 µm
Binding capacity Up to 300 µg per spin column Up to 300 µg per spin column
Gravity flow or spin column Spin column Spin column
Processing Automated/manual Automated
Scale Small scale Small scale
Special feature Low-throughput screening Up to 95% homogeneity in one step
Starting material Cell lysate Cell lysate
Support/matrix Macroporus silica Macroporus silica
Tag 6xHis tag 6xHis tag

Supporting data and figures

Publications

The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase.
Tafreshi NKh; Hosseinkhani S; Sadeghizadeh M; Sadeghi M; Ranjbar B; Naderi-Manesh H;
J Biol Chem; 2006; 282 (12):8641-7 2006 Dec 30 PMID:17197704
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
The promyelocytic leukemia protein functions as a negative regulator of IFN-gamma signaling.
Choi YH; Bernardi R; Pandolfi PP; Benveniste EN;
Proc Natl Acad Sci U S A; 2006; 103 (49):18715-20 2006 Nov 22 PMID:17121994
Stimulation of MCM helicase activity by a Cdc6 protein in the archaeon Thermoplasma acidophilum.
Haugland GT; Shin JH; Birkeland NK; Kelman Z;
Nucleic Acids Res; 2006; 34 (21):6337-44 2006 Nov 15 PMID:17108356

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
How do I prevent bubbles from forming in my Ni-NTA agarose column?
Gas bubbles may form when the resin undergoes a temperature change. To alleviate this problem, degas the Ni-NTA agarose in equilibration buffer or simply keep the agarose at a constant temperature to keep all the gases in solution.
FAQ ID -285
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
Can I use HEPES buffer instead of phosphate in my Ni-NTA column?
Although we do not strongly recommend it, up to 100 mM has been successfully used in some cases. Buffers that contain secondary or tertiary amines will reduce nickel ions and affect binding of 6xHis tagged proteins to the Ni-NTA resin.
FAQ ID -291
How does imidazole affect my quantitation of protein?

Since imidazole absorbs UV radiation at 280 nm, an elution profile measured at 280 nm while purifying a 6xHis tagged protein by FPLC will show an increase in absorbance above the background signal allowing quantitation of your protein. The absorbance of imidazole can vary depending on its source and purity, but elution buffer containing 250 mM imidazole usually has an A280 of 0.2–0.4.

To quantitate proteins in eluates containing imidazole, we recommend the Bradford protein assay, which is based on the ability of Coomassie Brilliant Blue R250 to form strong complexes with proteins. This assay is more tolerant of higher imidazole concentrations than the Lowry and the biuret assay, which are more sensitive to imidazole because they involve the reduction of copper. For accurate measurements, always use elution buffer without protein as the reagent blank.

FAQ ID -132
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 avoid poor immunolocalization morphology with Anti-His Antibodies?
Insufficient fixation of samples leads to poor morphological preservation. If using methanol or acetone try fixation with paraformaldehyde. If 2% paraformaldehyde is insufficient, increase fixative concentration to 4% and increase incubation time. Keep in mind that too harsh fixation can lead to functional alteration of the 6xHis epitope and loss of recognition by the Anti·His Antibodies.immunofluorescence
FAQ ID -200
How can I improve the expression of proteins containing hydrophobic regions?
The addition of either 1% Octylglycoside or 1% Dodecylmaltoside to the lysate might help to solubilize proteins containing large hydrophobic regions. The detergents should be added directly to the reaction mix.
FAQ ID -339
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
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