Ni-NTA Spin System

Hisタグ付加タンパク質の高速で小規模の精製向け

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Ni-NTA Spin Kit (50)

Cat. No. / ID: 31314

50 Ni-NTA Spin Columns、試薬、バッファー、コレクションチューブ、1 μgコントロール発現プラスミド

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￥81,000

KitColumn

Ni-NTA Spin Kit

Ni-NTA Spin Column

Ni-NTA Spin System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

カラム当たり最大300 μgのHisタグ付加タンパク質をわずか15分ほどで処理
非変性条件下や変性条件下での精製
ワンステップで最大95％の均一性
高速自動プロセッシングまたは手動プロセッシングのためのすぐ使用できるスピンカラム

Product Details

Ni-NTAシリカは、Ni-NTAを、非特異的な疎水性相互作用を抑制するために最適化されたマクロ多孔性のシリカ支持材料と組み合わせています。Ni-NTA Spin Kit内のNi-NTA Spin Columns（Hisタンパク質精製用スピンカラム）と別途入手できるNi-NTAシリカは便利なマイクロスピン形式で複数のサンプルを平行して容易に調製することが可能です。それらは、遺伝子工学的に製造されたタンパク質の機能的なスクリーニング、全長翻訳産物を発現するクローンの選択、発現レベルの比較のための簡単な方法を提供します。各スピンカラムは、最大300 µgまでのHisタグタンパク質を精製可能です。すべてのNi-NTAマトリックスと同様に、Ni-NTAスピンカラムは、自然な条件または変性条件下でのワンステップのタンパク質精製のために使用できます。Ni-NTA Spin Kitは、Hisタグタンパク質のスピン精製のための完全なキットで、QIAcube Connect 上で自動化することができます（画像「 QIAcube Connect」を参照）。

See figures

QIAcube Connect.

Performance

Ni-NTA Spin Columns（His-タンパク質精製用スピンカラム）はまた、Ni-NTA Spin Kitにも同梱されており、再現性の高い高速自動精製を（図 “再現性の高い自動精製”を参照）様々な異なる発現レベルで可能です（図 “異なる発現レベルでの精製”を参照）。

See figures

再現性の高い自動化精製。

さまざまな発現レベルでの精製

Principle

Ni-NTA Spin ColumnとNi-NTA Spin Kitを含むQIAexpress Ni-NTA Protein Purification Systemは、6個以上のヒスチジン残基のアフィニティータグ – 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）樹脂の顕著な選択性に基づいています。この技術により、非変性条件下または変性条件下でいかなる発現系からでもほぼあらゆるHisタグ付加タンパク質をワンステップで精製することが可能です。NTAはニッケルイオンに対して4つのキレーション部位を持っているため、金属イオンとの相互作用に利用できる3つの部位だけを持つ金属キレート精製システムよりもニッケルを強く結合します。余分なキレーション部位はニッケルイオンの浸出を防ぎ、その結果、より大きな結合能力を持つことができ、他の金属キレート精製システムを使用したものよりも高純度でタンパク質を精製することができます。QIAexpressシステムを使用して、バキュロウイルス、哺乳類細胞、酵母、細菌などのあらゆる発現系からHisタグタンパク質を精製できます。

Procedure

Hisタグタンパク質の精製は、4段階で構成されています：細胞溶解、結合、洗浄、溶出（図 “Ni-NTAタンパク質精製システムでのNi-NTA Spin Column精製”を参照）。QIAexpressシステムを使用した遺伝子組み換えタンパク質の精製は、タンパク質やHis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の、自然条件または変性条件下でのワンステップのタンパク質精製が可能になります。最大600 μlの細胞溶解物をNi-NTAスピンカラム上に充填できます。2分間のスピンでタグタンパク質はNi-NTAシリカに結合し、その一方で大部分のタグのないタンパク質は流れ出ます。洗浄段階の後、精製されたタンパク質は、温和な条件下（pH 5.9まで低下、または100～500 mMイミダゾールの添加など）で、100-300 μlの体積内に溶出します。Hisタグは小さく、ほぼ免疫性がないため、Hisタグの除去は通常は不要です。精製されたタンパク質は、すぐに使用できます。タンパク質は、複数の小規模な発現培地からはおよそ30分（手動）、またはおよそ60分（QIAcube Connectでの自動化）で精製することができます。強い変性剤や洗浄剤を使用して、レセプター、膜タンパク質、および封入体を形成するタンパク質を効率的に可溶化し精製できます。非特異的に結合する混入物の効率的な除去を可能にする試薬は、洗浄バッファーに含めることができます（表を参照）。精製されたタンパク質は、100～250 mMイミダゾールを競合剤として添加するか、pHを下げることにより温和な条件下で溶出します。

Ni-NTA–His相互作用に適合する試薬

6 M グアニジン塩酸塩
8 M 尿素
2% Triton X-100
2% Tween 20
1% CHAPS
20 mM β-ME
10 mM DTT
50% グリセロール
20% エタノール
2 M NaCl
4 M MgCl2
5 mM CaCl2
≤20 mM イミダゾール
20 mM TCEP

記載されている試薬は、指定されている濃度まで使用することができました。

See figures

Applications

Ni-NTA Spin ColumnとNi-NTA Spin Kitを含むQIAexpress Ni-NTA Protein Purification System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信頼性の高いワンステップのタンパク質精製を実現します。

構造的および機能的調査
3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相互作用を含むアッセイ
抗体を産生するための免疫付与

Ni-NTA Spin ColumnとNi-NTA Spin Kitの比較
機能	Ni-NTA Spin Column	Ni-NTA Spin Kit
アプリケーション	プロテオミクス	プロテオミクス
ビーズのサイズ	16–24 µm	16–24 µm
結合能	スピンカラム当たり最大300 µg	スピンカラム当たり最大300 µg
重力流またはスピンカラム	スピンカラム	スピンカラム
プロセッシング	自動化／手動	自動化
規模	小規模	小規模
特殊機能	ロースループットスクリーニング	ワンステップで最大95%の均一性
出発材料	細胞溶解物	細胞溶解物
支持体／マトリックス	マクロ多孔性シリカ	マクロ多孔性シリカ
タグ	6xHisタグ	6xHisタグ

Supporting data and figures

再現性の高い自動化精製。

示したタンパク質は、手動またはQIAcube上での自動化手順で、 5 ml LB培地由来のクリアなE. coli溶解物から、Ni-NTA Spin Columnを使用して非変性条件下で二重に精製した。CAT：クロラムフェ二コールアセチルトランスフェラーゼ、GFP：緑色蛍光タンパク質、HIV-RT：ヒト免疫不全ウイルス逆転写酵素、IL-1b：インターロイキン-1β。M：マーカー、C：クリアライセート（1レーン当たり2 μlを充填）、E：溶出分画（1レーン当たり3 μlを充填）。

Resources

Download Safety Data Sheets for QIAGEN product components.

For manual or automated purification of His-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

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

Gas bubbles may form when the resin undergoes a temperature change. To alleviate this problem, degas the Ni-NTA agarose in equilibration buffer or simply keep the agarose at a constant temperature to keep all the gases in solution.

FAQ ID -285

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

Although we do not strongly recommend it, up to 100 mM has been successfully used in some cases. Buffers that contain secondary or tertiary amines will reduce nickel ions and affect binding of 6xHis tagged proteins to the Ni-NTA resin.

FAQ ID -291

The Ni-NTA Spin columns used in the Ni-NTA Spin Kit contain a slightly different resin type than our other Ni-NTA columns. The columns in the Spin Kit contain approximately 25 mg of Ni-NTA Silica.

FAQ ID - 3354

Since imidazole absorbs UV radiation at 280 nm, an elution profile measured at 280 nm while purifying a 6xHis tagged protein by FPLC will show an increase in absorbance above the background signal allowing quantitation of your protein. The absorbance of imidazole can vary depending on its source and purity, but elution buffer containing 250 mM imidazole usually has an A₂₈₀ of 0.2–0.4.

To quantitate proteins in eluates containing imidazole, we recommend the Bradford protein assay, which is based on the ability of Coomassie Brilliant Blue R250 to form strong complexes with proteins. This assay is more tolerant of higher imidazole concentrations than the Lowry and the biuret assay, which are more sensitive to imidazole because they involve the reduction of copper. For accurate measurements, always use elution buffer without protein as the reagent blank.

FAQ ID -132

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

Insufficient fixation of samples leads to poor morphological preservation. If using methanol or acetone try fixation with paraformaldehyde. If 2% paraformaldehyde is insufficient, increase fixative concentration to 4% and increase incubation time. Keep in mind that too harsh fixation can lead to functional alteration of the 6xHis epitope and loss of recognition by the Anti·His Antibodies.immunofluorescence

FAQ ID -200

The addition of either 1% Octylglycoside or 1% Dodecylmaltoside to the lysate might help to solubilize proteins containing large hydrophobic regions. The detergents should be added directly to the reaction mix.

FAQ ID -339

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