Ni-NTA Superflow

Para la purificación de proteínas marcadas con 6xHis mediante FPLC

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Ni-NTA Superflow (25 ml)

Cat. No. / ID: 30410

Resina cargada de níquel de 25 ml (máx. presión: 140 psi)

Volume

25 mL

100 mL

500 mL

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✓ Procesamiento automático sin interrupción de pedidos en línea

✓ Servicio técnico y para productos experto y profesional

✓ Realización y repetición de pedidos rápidas y fiables

Características

Estabilidad mecánica superior, características destacables de flujo y alta capacidad de unión dinámica
Purificación práctica en un solo paso de proteínas marcadas con 6xHis
Permite tasas de flujo y presiones elevadas para aplicaciones eficientes a escala de producción y FPLC

Detalles del producto

Ni-NTA Superflow es una matriz de cromatografía de afinidad para la purificación de proteínas recombinantes portadoras de una etiqueta His.

Los residuos de histidina en la etiqueta His se unen a las posiciones vacantes en la esfera de coordinación de los iones de níquel inmovilizados con alta especificidad y afinidad. Los lisados celulares limpios se cargan en las matrices. Las proteínas marcadas con His se unen y otras proteínas pasan a través de la matriz. Tras lavarse, las proteínas marcadas con His se eluyen en un tampón en condiciones nativas o desnaturalizantes.

Rendimiento

Ni-NTA Superflow se compone de Ni-NTA combinado con resina Superflow. Combina una estabilidad mecánica superior con unas características destacables de flujo y una alta capacidad de unión dinámica. La capacidad para proteínas con etiquetas 6xHis es de 5–10 mg/mL. Esta resina permite la purificación en un solo paso de proteínas con etiquetas 6xHis utilizando tasas de flujo y presiones elevadas para aplicaciones eficientes a escala de producción y FPLC.

Principio

El QIAexpress Ni-NTA Protein Purification System se basa en la notable selectividad de la resina patentada Ni-NTA (ácido níquel-nitrilotriacético) para proteínas que contienen un marcador de afinidad de seis o más residuos de histidina: la etiqueta His. Esta tecnología permite la purificación en un paso de casi cualquier proteína marcada con His a partir de cualquier sistema de expresión en condiciones nativas o desnaturalizantes.

El NTA, que tiene cuatro lugares de quelación para los iones de níquel, une el níquel con mayor fuerza que los sistemas de purificación por quelación de metales que solo tienen tres lugares disponibles para la interacción con iones de metal. El lugar adicional de quelación impide la lixiviación iónica del níquel y da lugar a una mayor capacidad de unión y a preparaciones de proteína con una pureza más elevada que las obtenidas mediante otros sistemas de purificación por quelación de metales. Ni-NTA Superflow se puede utilizar para purificar proteínas marcadas con His a partir de cualquier sistema de expresión, incluyendo baculovirus, células de mamíferos, levaduras y bacterias.

Procedimiento

La purificación de las proteínas marcadas con His se divide en 4 etapas: lisis, unión, lavado y elución celular. La purificación de proteínas recombinantes utilizando el Ni-NTA Superflow no depende de la estructura tridimensional de la proteína o de la etiqueta 6xHis. Esto permite la purificación de proteínas en un paso en condiciones nativas o desnaturalizantes, a partir de soluciones diluidas y lisados sin procesar.

Los desnaturalizantes y detergentes fuertes se pueden utilizar para la solubilización y purificación eficientes de los receptores, las proteínas de membrana y las proteínas que forman cuerpos de inclusión. Los reactivos que permiten una eliminación eficiente de contaminantes de unión no específicos se pueden incluir en tampones de lavado (consulte la tabla).

Las proteínas purificadas se eluyen en condiciones suaves añadiendo 100–250 mM de imidazol como competidor o por reducción del pH.

Aplicaciones

Las matrices Ni-NTA proporcionan una purificación fiable en un solo paso de las proteínas marcadas con His adecuadas para cualquier aplicación, entre las que se incluyen:

Investigaciones estructurales y funcionales
Cristalización para la determinación de la estructura tridimensional
Ensayos de interacción proteína-proteína y proteína-ADN
Inmunización para producir anticuerpos
Aumento de la purificación a escala de producción

Para Ni-NTA Superflow, existe un expediente químico de fármaco (DMF) que contiene las características del producto, parámetros cromatográficos, información química y física, especificaciones de control de calidad, información sobre seguridad y toxicidad, y protocolos de purificación estándar para ayudar en cuestiones de regulación.

Datos y cifras de respaldo

Ni-NTA Superflow Cartridges

Para la purificación de proteína marcada con His utilizando sistemas de cromatografía líquida

Specifications

Features	Specifications
Applications	Proteómica
Scale	Gran escala
Processing	Manual
Bead size	60-160 µm
FPLC	Sí
Special feature	Purificación por lotes y columnas
Gravity flow or spin column	Flujo por gravedad o automatizado
Binding capacity	Hasta 50 mg/ml
Start material	Lisado celular
Support/matrix	Superflow
Tag	Etiqueta 6xHis
Yield	Depende de la capacidad de unión
Number of preps per run	1-24 muestras por serie

Recursos

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

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

A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.

Chen CS; Korobkova E; Chen H; Zhu J; Jian X; Tao SC; He C; Zhu H;

Nat Methods; 2007; 5 (1):69-74 2007 Dec 16 PMID:18084297

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

Calbindin D(9k) knockout mice are indistinguishable from wild-type mice in phenotype and serum calcium level.

Kutuzova GD; Akhter S; Christakos S; Vanhooke J; Kimmel-Jehan C; Deluca HF;

Proc Natl Acad Sci U S A; 2006; 103 (33):12377-81 2006 Aug 8 PMID:16895982

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

The Fast Start Columns in the QIAexpress Ni-NTA Fast Start Kit are prepacked with Ni-NTA Superflow resin.

FAQ ID -836

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

Yes, please follow either of the QIAGEN Supplementary Protocols:

FAQ ID -967

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

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

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