Ni-NTA Agarose

Zur Aufreinigung von Proteinen mit His-tag mittels Gravity-flow-Chromatographie

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Ni-NTA Agarose (25 ml)

Cat. No. / ID: 30210

25 ml mit Nickel beladenes Harz (max. Druck: 2,8 psi)

473,00 €

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Quantity

25 ml

100 ml

500 ml

Menge

Ni-NTA Agarose ist für molekularbiologische Anwendungen vorgesehen. Dieses Produkt ist nicht für die Diagnose, Prävention oder Behandlung einer Krankheit bestimmt.

✓ Automatische Verarbeitung von Online-Bestellungen 24/7

✓ Sachkundiger und professioneller technischer und Produkt-Support

✓ Schnelle und zuverlässige (Nach-)Bestellung

Eigenschaften

Ein-Schritt-Aufreinigung aus Rohlysat zu > 95 % reinem Protein
Hohe Bindungsaffinität und hohe Kapazität
Aufreinigung wahlweise unter nativen oder denaturierenden Bedingungen
Vorbeladene, gebrauchsfertige Matrizes für die Aufreinigung in jedem Maßstab
Automatisierte Aufreinigungs- und Assayprotokolle

Angaben zum Produkt

Ni-NTA-Agarose ist eine Affinitätschromatographie-Matrix zur Aufreinigung rekombinanter Proteine, die einen His-tag tragen. Histidin-Reste im His-tag binden mit hoher Spezifität und Affinität an die freien Positionen der Koordinationssphäre der immobilisierten Nickelionen. Die geklärten Zelllysate werden auf die Matrizes geladen. Die Proteine mit His-tag werden gebunden, und andere Proteine passieren die Matrix. Nach dem Waschen werden die Proteine mit His-tag unter nativen oder denaturierenden Bedingungen in Puffer eluiert.

Leistung

Ni-NTA-Agarose besteht aus Ni-NTA, das an einen Sepharose CL-6B-Träger gekoppelt ist, und eine hohe Bindekapazität bei minimaler unspezifischer Bindung bietet (siehe Abbildung Ein-Schritt-Aufreinigung unter nativen Bedingungen). Dieses Material lässt sich für die meisten Maßstäbe von Batch- und Säulenaufreinigungsverfahren ausgezeichnet handhaben. Ni-NTA-Agarose ist einzeln oder als Komponente der QIAexpress Kits verfügbar. Hierbei handelt es sich um vollständige Kits für die effiziente Expression und Aufreinigung von Proteinen mit His-tag aus E. coli.

Abbildungen ansehen

Ein-Schritt-Aufreinigung unter nativen Bedingungen.

Prinzip

Das QIAexpress Ni-NTA Protein Purification System basiert auf der bemerkenswerten Selektivität des patentierten Ni-NTA(Nickel-Nitrilotriessigsäure)-Harzes für Proteine mit einem Affinitätstag aus sechs oder mehr Histidinresten (zusammenhängend oder alternierend) – dem His-tag. Diese Technologie erlaubt die Ein-Schritt-Aufreinigung nahezu jedes Proteins mit His-tag aus jedem Expressionssystem unter nativen oder denaturierenden Bedingungen. NTA, das über vier Chelationen-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 für die Aufreinigung von Proteinen mit His-tag aus jedem Expressionssystem, einschließlich Baculoviren, Säugerzellen, Hefen und Bakterien verwendet werden.

Verfahren

Die Aufreinigung von mit His-tag markierten Proteinen besteht aus 4 Phasen: Zelllyse, Bindung, Waschen und Elution (siehe ). Die Aufreinigung rekombinanter Proteine mit dem QIAexpress System ist nicht von der dreidimensionalen Struktur des Proteins oder des 6xHis-tag abhängig. Dadurch ist eine Ein-Schritt-Proteinaufreinigung sowohl unter nativen als auch unter denaturierenden Bedingungen, ausgehend von verdünnten Lösungen und Rohlysaten, möglich. 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 His/Ni-NTA-Interaktion kompatible Reagenzien
Denaturierende Agenzien	Detergenzien	Reduzierende Agenzien	Sonstige	Salze	Für die langfristige Lagerung
6 M Gu-HCl	2 % Triton X-100	20 mM β-ME	50 % Glycerin	4 M MgCl₂	Bis zu 30 % Ethanol
8 M Harnstoff	2 % Tween 20	10 mM DTT	20 % Ethanol	5 mM CaCl₂	oder 100 mM NaOH
	1 % CHAPS	20 mM TCEP	20 mM Imidazol	2 M NaCl

Abbildungen ansehen

Proteinaufreinigung mit dem Ni-NTA-Proteinaufreinigungssystem.

Anwendungen

Ni-NTA-Matrizes ermöglichen eine zuverlässige Ein-Schritt-Aufreinigung von Proteinen mit His-tag, die für jede nachfolgende Applikation geeignet sind, einschließlich:

Struktur- und Funktionsuntersuchungen
Kristallisierung zur Bestimmung der dreidimensionalen Struktur
Protein-Protein- und Protein-DNA-Interaktionsassays
Immunisierung zur Antikörperproduktion
Aufskalierung der Aufreinigung auf Produktionsmaßstab

Ergänzende Daten und Abbildungen

Proteinaufreinigung mit dem Ni-NTA-Proteinaufreinigungssystem.

Specifications

Features	Specifications
Applications	Proteomik
Tag	6xHis-tag
Bead size	45–165 µm
Special feature	Batch- und Säulenaufreinigung
FPLC	Nein
Processing	Manuell
Support/matrix	Sepharose CL-6B
Start material	Zelllysat
Scale	Großer Maßstab
Binding capacity	Bis zu 50 mg/ml
Gravity flow or spin column	Gravity flow
Yield	100 µg–100 mg

Ressourcen

A handbook for high-level expression and purification of 6xHis-tagged proteins

Publications

Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion.

Aizen J; Kasuto H; Golan M; Zakay H; Levavi-Sivan B;

Biol Reprod; 2006; 76 (4):692-700 2006 Dec 27 PMID:17192515

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

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

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

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

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

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

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

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

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

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 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

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

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