Ni-NTA Fast Start Kit

Para la purificación y detección de proteínas recombinantes marcadas con His a partir de lisados de E. coli

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Ni-NTA Fast Start Kit (6)

Cat. No. / ID: 30600

Para la purificación y detección de seis preparaciones de proteína marcadas con 6xHis: 6 x Fast Start Columns, Penta·His Antibody, Buffers y Reagents

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El Ni-NTA Fast Start Kit está concebido para su uso en aplicaciones de biología molecular. Este producto no está concebido para el diagnóstico, la prevención ni el tratamiento de enfermedades.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

Ideal para los investigadores que se inician en la ciencia de proteínas
Todo lo necesario para una purificación eficiente en un solo kit
Protocolos fáciles de seguir y procesamiento sencillo
Hasta 25 mg de proteína por columna en tan solo 90 minutos

Product Details

El Ni-NTA Fast Start Kit proporciona todo lo necesario para una purificación eficiente y rápida de proteínas marcadas con His a partir de lisados limpios de E.coli, incluyendo columnas de Ni-NTA precargadas. Los tampones suministrados en el kit permiten que las proteínas se purifiquen en condiciones nativas o desnaturalizantes. El kit también contiene un anticuerpo Anti-His para la detección de proteínas expresadas marcadas con His.

Performance

El Ni-NTA Fast Start Kit proporciona todo lo necesario para una purificación eficiente y rápida de proteínas marcadas con His a partir de lisados limpios de E. coli. Los tampones suministrados en el kit permiten que las proteínas se purifiquen en condiciones nativas o desnaturalizantes (consulte la figura ) Los protocolos sencillos y simples y los reactivos preparados para usar hacen que el Ni-NTA Fast Start Kit sea un punto de inicio ideal para los científicos que buscan ampliar su investigación en el campo de la expresión de proteínas, pero no están familiarizados con los procedimientos de purificación de proteínas.

See figures

Proteínas de gran pureza en condiciones nativas o desnaturalizantes.

Principle

El Ni-NTA Fast Start Kit se basa en la selectividad notable de la resina patentada de Ni-NTA (ácido níquel-nitrilotriacético) para proteínas que contienen un marcador de afinidad de seis o más residuos de histidina (consecutivo o alterno): el marcador His. Esta tecnología permite la purificación en un paso de casi cualquier proteína marcada con His 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 preparaciones de proteína con una pureza más elevada que las obtenidas mediante otros sistemas de purificación por quelación de metales. El marcador His también forma el epítopo que reconoce el Penta His Antibody suministrado con el kit.

Procedure

Las células se lisian en el tampón de lisis suministrado y se centrifugan para obtener un lisado limpio. El lisado se aplica a una Fast Start Column, donde las proteínas marcadas con His se unen con gran afinidad y las proteínas sin marcar pasan a través de esta. Tras lavarse, las proteínas purificadas se eluyen utilizando tampones que contienen imidazol (condiciones nativas) o de bajo pH (condiciones desnaturalizantes) (consulte la figura ). El procedimiento de purificación y el rendimiento de proteínas pueden ir seguidos de SDS-PAGE, western blotting e inmunodetección con el Penta·His Antibody suministrado.

See figures

Proteína de gran pureza marcada con His.

Applications

El Ni-NTA Fast Start Kit proporciona una purificación fiable en un paso de proteínas marcadas son 6xHis adecuadas para cualquier aplicación, donde se incluyen:

Estudios funcionales
Inmunización para producir anticuerpos
Ensayos relacionados con interacciones proteína-proteína y proteína-ADN
Investigaciones estructurales

Supporting data and figures

Proteína de gran pureza marcada con His.

Specifications

Features	Specifications
Applications	Proteómica
Support/matrix	Matriz Ni-NTA
Processing	Manual
Binding capacity	5-20 mg/ml
Special feature	Tecnología Ni-NTA
Gravity flow or spin column	Flujo de gravedad
Start material	Lisado celular
Tag	Etiqueta 6xHis
Yield	<10 mg de proteína por columna

Resources

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

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 composition of the elution buffer in the Ni-NTA Fast Start Kit is 50 mM Na-phosphate, 300 mM NaCl and 250 mM imidazole at pH 8.0.

FAQ ID - 3353

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

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