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Ni-NTA Spin Kit (50)

Cat. No. / ID:  31314

50 Ni-NTA-Spin-Säulen, Reagenzien, Puffer, Auffangröhrchen, 1 μg Kontroll-Expressionsplasmid
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Ni-NTA Spin Kit
Ni-NTA Spin Column
Ni-NTA Spin System sind für molekularbiologische Anwendungen vorgesehen. Diese Produkte sind nicht zur Diagnose, Prävention oder Behandlung einer Erkrankung vorgesehen.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Bis zu 300 μg Protein mit His-tag pro Säule in nur 15 Minuten
  • Aufreinigung unter nativen und denaturierenden Bedingungen
  • Bis zu 95 % Homogenität in einem Schritt
  • Gebrauchsfertige Spin-Säulen für die schnelle automatische oder manuelle Verarbeitung

Product Details

Ni-NTA-Silica kombiniert Ni-NTA mit einem makroporösen Siliziumdioxid-Trägermaterial, das zur Vermeidung unspezifischer hydrophober Wechselwirkungen optimiert wurde. Ni-NTA Spin Columns (Spin-Säulen zur Aufreinigung von His-Proteinen), die im Ni-NTA Spin Kit enthalten und auch separat erhältlich sind, bieten Ni-NTA-Silica in einem praktischen Mikrospin-Format für die einfache und parallele Präparation mehrerer Proben. Sie bieten ein einfaches Verfahren zum funktionellen Screening von gentechnisch hergestellten Proteinen, zur Selektion von Klonen, die Translationsprodukte in voller Länge exprimieren, und zum Vergleich von Expressionsniveaus. Jede Spin-Säule kann bis zu 300 µg Protein mit His-tag aufreinigen. Wie alle Ni-NTA-Matrizes können Ni-NTA-Spin-Säulen für die Ein-Schritt-Proteinaufreinigung unter nativen und denaturierenden Bedingungen genutzt werden. Das Ni-NTA Spin Kit ist ein Komplettkit für die Spin-Säulen-Aufreinigung von Proteinen mit His-tag. Es kann auf dem QIAcube Connect automatisiert werden (siehe Abbildung „QIAcube Connect“).

Performance

Ni-NTA Spin Columns (Spin-Säulen zur Aufreinigung von His-Proteinen), die ebenfalls im Ni-NTA Spin Kit enthalten sind, ermöglichen eine reproduzierbare, schnelle und automatisierte Aufreinigung (siehe Abbildung  “Reproduzierbare, automatisierte Aufreinigung”) bei unterschiedlichen Expressionsniveaus (siehe Abbildung  “Aufreinigung bei unterschiedlichen Expressionsniveaus”).

See figures

Principle

Das QIAexpress Ni-NTA-System zur Proteinaufreinigung, einschließlich der Ni-NTA Spin Columns und des Ni-NTA Spin Kits, basiert auf der bemerkenswerten Selektivität des patentierten Nickel-Nitrilotriessigsäure(Ni-NTA)-Harzes für Proteine, die einen Affinitäts-Tag mit sechs oder mehr Histidinresten – einen His-tag – aufweisen. Diese Technologie ermöglicht eine Ein-Schritt-Aufreinigung fast aller Proteine mit His-tag aus jedem Expressionssystem unter nativen oder denaturierenden Bedingungen. NTA, das über vier Chelation-Bindestellen für Nickelionen verfügt, bindet Nickel fester als metallchelatbildende Aufreinigungssysteme, die nur drei verfügbare Bindestellen für die Interaktion mit Metallionen besitzen. Die zusätzliche Chelation-Bindestelle verhindert das Auswaschen von Nickelionen und führt zu einer höheren Bindungskapazität und damit zu Proteinpräparationen mit höherer Reinheit verglichen mit denen, die mit anderen metallchelatbildenden Aufreinigungssystemen gewonnen wurden. Das QIAexpress System kann zur Aufreinigung von Proteinen mit His-tag aus allen Expressionssystemen, einschließlich Baculovirus, Säugerzellen, Hefen und Bakterien, genutzt werden.

Procedure

Die Aufreinigung von Proteinen mit His-tag besteht aus 4 Phasen: Zelllyse, Bindung, Waschen und Elution (siehe Abbildung “Ni-NTA-Spin-Säulen-Aufreinigung mit dem Ni-NTA-Proteinaufreinigungssystem”). Die Aufreinigung rekombinanter Proteine mit dem QIAexpress System ist nicht von der dreidimensionalen Struktur des Proteins oder des His-tags abhängig. Dadurch ist eine Ein-Schritt-Proteinaufreinigung sowohl unter nativen als auch unter denaturierenden Bedingungen, ausgehend von verdünnten Lösungen und Rohlysaten, möglich. Bis zu 600 μl eines Zelllysats werden in eine Ni-NTA-Spin-Säule geladen. Ein schneller zweiminütiger Spin bindet das markierte Protein an das Ni-NTA-Silica, während die meisten nicht markierten Proteine durchlaufen. Nach einem Waschschritt wird das aufgereinigte Protein unter milden Bedingungen (z. B. Absenkung des pH-Wertes auf 5,9 oder Zugabe von 100–500 mM Imidazol) in einem Volumen von 100–300 μl eluiert. In der Regel muss der His-tag nicht entfernt werden, da er klein und meist nicht immunogen ist. Das aufgereinigte Protein ist sofort einsatzbereit. Proteine können aus mehreren kleinen Expressionskulturen in etwa 30 Minuten (manuelles Verfahren) oder in etwa 60 Minuten (automatisiert mit QIAcube Connect) aufgereinigt werden. Starke denaturierende Agenzien und Detergenzien können für eine effiziente Solubilisierung und Aufreinigung von Rezeptoren, Membranproteinen und Proteinen, die Einschlusskörper (inclusion bodies) bilden, eingesetzt werden. Reagenzien, die eine effiziente Entfernung unspezifisch bindender Kontaminanten ermöglichen, können den Waschpuffern hinzugefügt werden (siehe Tabelle). Die aufgereinigten Proteine werden unter milden Bedingungen durch Zugabe von 100–250 mM Imidazol als Kompetitor oder durch Absenkung des pH-Werts eluiert.

Mit der Ni-NTA–His-Wechselwirkung kompatible Reagenzien:

  • 6 M Guanidin-HCl
  • 8 M Harnstoff
  • 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 Imidazol
  • 20 mM TCEP
See figures

Applications

Das QIAexpress Ni-NTA-System zur Proteinaufreinigung, einschließlich der Ni-NTA Spin Columns und des Ni-NTA Spin Kits, ermöglicht eine zuverlässige Ein-Schritt-Aufreinigung von Proteinen, die sich für jede Anwendung eignen wie:

  • Struktur- und Funktionsuntersuchungen
  • Kristallisierung zur Bestimmung der dreidimensionalen Struktur
  • Protein-Protein- und Protein-DNA-Interaktionsassays
  • Immunisierung zur Antikörperproduktion

 

Vergleich der Ni-NTA Spin Columns und des Ni-NTA Spin Kits
Eigenschaften Ni-NTA Spin Columns Ni-NTA Spin Kit
Anwendungen Proteomik Proteomik
Bead-Größe 16–24 µm 16–24 µm
Bindekapazität Bis zu 300 µg je Spin-Säule Bis zu 300 µg je Spin-Säule
Gravity-flow oder Spin-Säule Spin-Säule Spin-Säule
Verarbeitung Automatisiert/manuell Automatisiert
Maßstab Kleiner Maßstab Kleiner Maßstab
Besondere Eigenschaften Screening mit niedrigem Durchsatz Bis zu 95 % Homogenität in einem Schritt
Ausgangsmaterial Zelllysat Zelllysat
Träger/Matrix Makroporöses Siliziumdioxid (Silica) Makroporöses Siliziumdioxid (Silica)
Tag 6xHis-tag 6xHis-tag

Supporting data and figures

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

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
3354 - What type and amount of resin is packed into the Ni-NTA Spin columns from the Ni-NTA Spin Kit?

The Ni-NTA Spin columns used in the Ni-NTA Spin Kit contain a slightly different resin type than our other Ni-NTA columns. The columns in the Spin Kit contain approximately 25 mg of Ni-NTA Silica.

FAQ ID - 3354
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