Ni-NTA Agarose

自然落下クロマトグラフィーによるHisタグ付きタンパク質の精製用

S_0276_PROT_niNTA

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Ni-NTA Agarose (25 ml)

Cat. No. / ID:  30210

25 mlニッケルチャージ樹脂(最大圧力:2.8 psi)
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$435.00
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Quantity
25 ml
100 ml
500 ml
Ni-NTA Agaroseは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 粗溶解物から純度95%を上回るタンパク質をワンステップ精製
  • 高い結合親和性と高容量
  • 天然条件下または変性条件下での精製の選択
  • どのような精製規模にも対応可能な事前チャージ済みのすぐに使用可能なマトリックス
  • 自動精製およびアッセイプロトコール

Product Details

Ni-NTA Agaroseは、Hisタグを持つ組み換えタンパク質を精製するためのアフィニティークロマトグラフィーマトリックスです。Hisタグのヒスチジン残基は、高い特異性と親和性で、固定化ニッケルイオンの配位圏の空いた位置に結合します。澄明な細胞溶解物をマトリックスにロードします。Hisタグ付きタンパク質は結合し、他のタンパク質はマトリックスを通過します。洗浄後、Hisタグ付きタンパク質は、天然または変性条件下でバッファーに溶出されます。

Performance

Ni-NTA AgaroseはSepharose CL-6Bサポートに結合したNi-NTAを提供し、結合能は高く、非特異的結合は最小限です(図  天然条件下でのワンステップ精製を参照)。この物質は、ほとんどの規模のバッチ精製とカラム精製に優れた取扱特性を示します。Ni-NTA Agaroseは個別でも、E. coliからHisタグ付きタンパク質を効率的に発現および精製するための完全なキットであるQIAexpress Kitのコンポーネントとしてもご利用いただけます。

See figures

Principle

QIAexpress Ni-NTAタンパク質精製システムは、6個以上のヒスチジン残基(連続または交互)のアフィニティタグ(Hisタグ)を含むタンパク質に対する特許取得済みのNi-NTA(ニッケル-ニトリロ三酢酸)樹脂の優れた選択性に基づいています。この技術により、天然または変性条件下で、あらゆる発現系から、ほぼすべてのHisタグ付きタンパク質をワンステップ精製できます。ニッケルイオンに対して4つのキレート部位を持つNTAは、金属イオンとの相互作用に利用できる部位が3つしかない金属キレート精製システムよりも強くニッケルに結合します。余剰なキレート部位がニッケルイオンの浸出を防ぐため、他の金属キレート精製システムを使用した場合よりも結合能が高く、高純度のタンパク質調製物が得られます。QIAexpressシステムを使用すると、バキュロウイルス、哺乳類細胞、酵母、細菌など、あらゆる発現系からHisタグ付きタンパク質を精製できます。

Procedure

Hisタグ付きタンパク質の精製は、細胞溶解、結合、洗浄、溶出の4段階で構成されています( Ni-NTA タンパク質精製システムによるタンパク質精製を参照)。QIAexpressシステムを用いる組み換えタンパク質の精製は、タンパク質や6xHisタグの3次元構造に依存しません。これにより、希釈溶液や粗溶解物から、天然または変性条件下で、タンパク質をワンステップ精製できます。受容体、膜タンパク質、封入体を形成するタンパク質の効率的な可溶化と精製には、強力な変性剤や洗浄剤を使用できます。非特異的に結合する汚染物質を効率的に除去できる試薬を洗浄バッファーに含めることができます(表を参照)。精製したタンパク質は、競合剤として100~250 mMのイミダゾールを加えるか、pHを下げることにより、穏やかな条件下で溶出します。

His/Ni-NTA相互作用に対応する試薬
変性剤界面活性剤還元剤その他長期保存用
6 M Gu·HCl2% Triton X-10020 mM β-ME50%グリセロール4 M MgCl2最大30%エタノール
8 M尿素2% Tween 2010 mM DTT20%エタノール5 mM CaCl2または100 mM NaOH
1% CHAPS20 mM TCEP20 mMイミダゾール2 M NaCl
See figures

Applications

Ni-NTAマトリックスは、以下をはじめとするあらゆるアプリケーションに適したHisタグ付きタンパク質を、高い信頼性でワンステップ精製します。

  • 構造および機能研究
  • 三次元構造決定のための結晶化
  • タンパク質-タンパク質およびタンパク質-DNA相互作用アッセイ
  • 抗体を産生するための免疫付与
  • 精製を生産規模にスケールアップ

Supporting data and figures

Specifications

FeaturesSpecifications
Applicationsプロテオミクス
Tag6xHisタグ
Bead size45~165 µm
特殊機能バッチおよびカラム精製
FPLCいいえ
Processing手動
Support/matrixSepharose CL-6B
Start material細胞溶解物
Scale大規模
Binding capacity最大50 mg/ml
Gravity flow or spin column自然落下
Yield100 µg~100 mg

Resources

MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
キットハンドブック (2)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Certificates of Analysis (1)
Kit Handbooks (2)
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
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

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
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
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
What is the difference between Ni-NTA Agarose and Ni-NTA Superflow?

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
Can I reuse the Ni-NTA Agarose and Ni-NTA Superflow resins?

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
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 check if any residual proteins remain on the Ni-NTA Agarose matrix after elution?
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
3351 - What is the upper limit for protein size that can bind to Ni-NTA agarose resin?
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
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
3352 - What are the size ranges of Ni-NTA particles?
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
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