Ni-NTA Fast Start Kit

Pour la purification et la détection des protéines recombinantes marquées à l’histidine (His) à partir de lysats d’E. coli

S_0253_PROT_NiNTA067

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Ni-NTA Fast Start Kit (6)

Cat. No. / ID:  30600

Pour la purification et la détection de six préparations de protéines marquées à l’hexahistidine (6xHis) : 6 × colonnes Fast Start, anticorps anti-pentahistidine, tampons et réactifs
Copy order details
498,00 €
Log in To see your account pricing.
Le Ni-NTA Fast Start Kit est destiné aux applications de biologie moléculaire. Ce produit n’est pas conçu pour le diagnostic, la prévention ou le traitement des maladies.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Idéal pour les chercheurs qui débutent avec les protéines
  • Tous les éléments nécessaires à une purification performante dans un kit
  • Protocoles faciles à suivre et traitement simple
  • Jusqu’à 25 mg de protéines par colonne en 90 minutes seulement

Product Details

Le Ni-NTA Fast Start Kit contient tout ce qu’il faut pour une purification rapide et performante des protéines marquées à l’histidine (His) à partir de lysats d’E.coli clarifiés, y compris des colonnes Ni-NTA préremplies. Les tampons du kit permettent de purifier les protéines en conditions natives ou dénaturantes. Le kit contient également un anticorps anti-His pour la détection des protéines marquées à l’histidine (His) exprimées.

Performance

Le Ni-NTA Fast Start Kit contient tout ce qu’il faut pour une purification rapide et performante des protéines marquées à l’histidine (His) à partir de lysats d’E.coli clarifiés. Les tampons du kit permettent de purifier les protéines en conditions natives ou dénaturantes (voir l’illustration  Protéines de grande pureté en conditions natives ou dénaturantes). Avec ses protocoles simples et faciles et ses réactifs prêts à l’emploi, le Ni-NTA Fast Start Kit est le point de départ idéal pour les scientifiques qui souhaitent élargir leur domaine de recherche à l’expression des protéines, mais qui ne maîtrisent pas encore les procédures de purification des protéines.
See figures

Principle

Le Ni-NTA Fast Start Kit est basé sur la remarquable sélectivité de la résine brevetée Ni-NTA (Nickel-Acide nitrilotriacétique) pour les protéines contenant une étiquette d’affinité de six résidus d’histidine (consécutifs ou alternatifs) ou plus, le His-tag. Cette technologie permet une purification en une étape de quasiment toutes les protéines marquées à l’histidine en conditions natives ou dénaturantes. Le NTA, qui présente quatre sites de chélation pour les ions nickel, lie le nickel plus étroitement que les systèmes de purification par chélation du métal, qui ne présentent que trois sites pour l’interaction avec les ions métalliques. Le site de chélation supplémentaire empêche la lixiviation des ions nickel et offre des préparations des protéines d’une pureté supérieure à celle obtenue avec d’autres systèmes de purification par chélation du métal. Le His-tag forme aussi l’épitope reconnu par l’anticorps anti-pentahistidine présent dans le kit.

Procedure

Les cellules sont lysées dans le tampon de lyse fourni puis centrifugées afin d’obtenir un lysat clarifié. Ce lysat est appliqué à une colonne Fast Start, dans laquelle les protéines marquées à l’histidine (His) se lient avec une affinité forte tandis que les protéines non marquées traversent. Après le lavage, les protéines purifiées sont éluées à l’aide de tampons contenant de l’imidazole (conditions natives) ou de faible pH (conditions dénaturantes) (voir l’illustration  Protéines infiniment pures marquées à l’hexahistidine (6xHis)). Après la procédure de purification et le rendement des protéines, vous pouvez utiliser la SDS-PAGE, le transfert de western et l’immunodétection avec l’anticorps anti-pentahistidine fourni.
See figures

Applications

Le Ni-NTA Fast Start Kit permet une purification fiable en une étape des protéines marquées à l’hexahistidine (6xHis), adaptée à de nombreuses applications, notamment :

  • Étude des fonctionnements
  • Immunisation aux anticorps produits
  • Dosages impliquant des interactions protéine–protéine et protéine–ADN
  • Étude des structures

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsProtéomique
Support/matrixMatrice Ni-NTA
ProcessingManuel
Binding capacity5 à 20 mg/ml
Special featureTechnologie Ni-NTA
Gravity flow or spin columnGravité
Start materialLysat cellulaire
Tag6xHis-tag
Yield< 10 mg de protéine par colonne

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
Which resin is used in the QIAexpress Ni-NTA Fast Start Columns?
The Fast Start Columns in the QIAexpress Ni-NTA Fast Start Kit are prepacked with Ni-NTA Superflow resin.
FAQ ID -836
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
Should I use Ni-NTA Agarose in column or batch format for purification of 6xHis-tagged proteins?
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
3353 - What is the composition of the elution buffer in the Ni-NTA Fast Start Kit?

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
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 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