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Ni-NTA Spin Kit (50)

Cat. No. / ID:  31314

50 个 Ni-NTA 离心柱,试剂,缓冲液,采样管,1 μg 对照表达质粒
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Ni-NTA Spin Kit
Ni-NTA Spin Column
Ni-NTA Spin System 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 每个离心柱可在短短 15 分钟内纯化多达 300 μg His 标签蛋白
  • 在天然和变性条件下进行纯化
  • 一步实现高达 95% 的均一性
  • 用于快速自动化或手动处理的即用型离心柱

Product Details

Ni-NTA 硅胶将 Ni-NTA 与大孔硅胶载体材料相结合,并经过优化以抑制非特异性疏水相互作用。Ni-NTA Spin Kit 内附带以及可单独供应的 Ni-NTA Spin Column(His 蛋白纯化离心柱)提供了便利的微离心形式的 Ni-NTA 硅胶,能够很容易地同时制备多个样本。它们为工程化蛋白的功能筛选、表达全长翻译产物的克隆选择以及表达水平的比较提供了一种简单的方法。每个离心柱可以纯化多达 300 µg His 标签蛋白。与所有 Ni-NTA 基质一样,Ni-NTA 离心柱可用于在天然或变性条件下的一步法蛋白质纯化。Ni-NTA Spin Kit 是用于 His 标签蛋白离心纯化的完整试剂盒。它可以在 QIAcube Connect 上实现自动化(参见图片“ QIAcube Connect”)。

See figures

Performance

Ni-NTA Spin Column(His 蛋白纯化离心柱)也包含在 Ni-NTA Spin Kit 中,允许在不同表达水平下(参见图片 “不同表达水平下的纯化”)进行可重复的快速自动纯化(参见图片 “可重复的自动纯化”)。

See figures

Principle

QIAexpress Ni-NTA Protein Purification System,包括 Ni-NTA Spin Columns 和 Ni-NTA Spin Kit,基于专利 Ni-NTA(镍-次氮基三乙酸)树脂,它对含有六个或更多组氨酸残基(即 His 标签)的亲和标签的蛋白质具有显著的选择性。该技术允许在天然或变性条件下从任何表达系统中一步纯化几乎任何 His 标签蛋白。NTA 具有四个镍离子螯合位点,与只有三个可用于与金属离子相互作用位点的金属螯合纯化系统相比,其与镍的结合更加紧密。这个额外的螯合位点能够防止镍离子浸出,与其他金属螯合纯化系统相比,能够产生更强的结合能力并得到更高纯度的蛋白质制剂。QIAexpress 系统可用于从包括杆状病毒、哺乳动物细胞、酵母和细菌在内的任何表达系统中纯化 His 标签蛋白。

Procedure

His 标签蛋白的纯化包括 4 个步骤:细胞裂解、结合、洗涤和洗脱(参见图片“使用 Ni-NTA 蛋白纯化系统进行 Ni-NTA 离心柱纯化”)。使用 QIAexpress 系统进行重组蛋白纯化不依赖于蛋白质或 His 标签的三维结构。这允许在天然或变性条件下从稀释溶液和粗制裂解物中一步纯化蛋白质。将最多 600 μl 细胞裂解物装载至 Ni-NTA 离心柱。快速离心 2 分钟即可使标签蛋白结合至 Ni-NTA 硅胶,而大多数非标签蛋白都会流过硅胶。经过一次洗涤步骤后,在温和条件下(例如 pH 降低至 5.9,或加入 100-500 mM 咪唑)以 100-300 μl 的体积洗脱纯化的蛋白质。通常不需要去除 His 标签,因为它很小,而且很少具有免疫原性。纯化后的蛋白质可以立即使用。可在大约 30 分钟(手动程序)或大约 60 分钟(QIAcube Connect 自动化程序)内从多种小规模表达培养物中纯化出蛋白质。可使用强变性剂和洗涤剂进行受体、膜蛋白和形成包涵体的蛋白质的高效溶解和纯化。洗涤缓冲液中可以包括能够高效去除非特异性结合污染物的试剂(参见表格)。通过加入 100-250 mM 咪唑作为竞争物或通过降低 pH 在温和条件下洗脱纯化的蛋白质。

与 Ni-NTA–His 相互作用兼容的试剂:

  • 6 M 盐酸胍
  • 8 M 尿素
  • 2% Triton X-100
  • 2% Tween 20
  • 1% CHAPS
  • 20 mM β-ME
  • 10 mM DTT
  • 50% 甘油
  • 20% 乙醇
  • 2 M NaCl
  • 4 M MgCl2
  • 5 mM CaCl2
  • ≤20 mM 咪唑
  • 20 mM TCEP
See figures

Applications

QIAexpress Ni-NTA Protein Purification System,包括 Ni-NTA Spin Columns 和 Ni-NTA Spin Kit,提供了适用于任何应用的可靠一步法蛋白质纯化,包括:

  • 结构和功能研究
  • 结晶用于三维结构测定
  • 涉及蛋白质–蛋白质和蛋白质–DNA 相互作用的检测
  • 免疫接种产生抗体

 

Ni-NTA Spin Columns 与 Ni-NTA Spin Kit 的比较
特点 Ni-NTA Spin Columns Ni-NTA Spin Kit
应用 蛋白质组学 蛋白质组学
微珠大小 16–24 µm 16–24 µm
结合能力 每个离心柱最多 300 µg 每个离心柱最多 300 µg
重力流或离心柱 离心柱 离心柱
处理 自动化/手动 自动化
规模 小规模 小规模
特殊特点 低通量筛选 一步实现高达 95% 的均一性
起始材料 细胞裂解物 细胞裂解物
载体/基质 大孔硅胶 大孔硅胶
标签 6xHis 标签 6xHis 标签

Supporting data and figures

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

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
How do I prevent bubbles from forming in my Ni-NTA agarose column?
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
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
Can I use HEPES buffer instead of phosphate in my Ni-NTA column?
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
3354 - What type and amount of resin is packed into the Ni-NTA Spin columns from the Ni-NTA Spin Kit?

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
How does imidazole affect my quantitation of protein?

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
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 avoid poor immunolocalization morphology with Anti-His Antibodies?
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
How can I improve the expression of proteins containing hydrophobic regions?
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
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
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