S_0276_PROT_niNTA

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Ni-NTA Agarose (25 ml)

Cat. No. / ID:  30210

25 ml 镍结合树脂(最大压力:2.8 psi)
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25 ml
100 ml
500 ml
Ni-NTA Agarose 旨在用于分子生物学应用。该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

  • 从粗裂解物中一步纯化出纯度大于 95% 的蛋白
  • 高结合亲和力和高容量
  • 非变形或变性条件下的纯化选择
  • 预充即用型基质,适用于任何规模的纯化
  • 自动纯化和检测方案

Product Details

Ni-NTA Agarose 是一种亲和层析基质,用于纯化带有 His 标签的重组蛋白。His 标签中的组氨酸残基凭借高特异性和亲和力,与固定镍离子配位球中的空位结合。澄清的细胞裂解物被加载到基质上。His 标签蛋白与基质结合,其他蛋白则穿过基质。洗涤后,在非变性或变性条件下在缓冲液中洗脱 His 标签蛋白。

Performance

Ni-NTA Agarose 将 Ni-NTA 与 Sepharose CL-6B 支链耦合在一起,具有高结合能力,非特异性结合极少(见图 原生条件下一步纯化)。这种材料具有出色的处理性能,适用于大多数规模的间歇式和柱式纯化。Ni-NTA Agarose 既可单独提供,也可作为 QIAexpress Kits 的组成部分提供,这些试剂盒是用于从大肠杆菌中高效表达和纯化 His 标签蛋白的全套试剂盒。

See figures

Principle

QIAexpress Ni-NTA 蛋白纯化系统基于的是获得专利的 Ni-NTA(次氮基三乙酸镍)树脂对含有六个或更多组氨酸残基(连续或交替)的亲和标记(His 标记)的蛋白的显著选择性。该技术可在非变性或变性条件下,从任何表达系统中一步纯化几乎所有 His 标签蛋白。NTA 有四个镍离子螯合位点,与只有三个位点可与金属离子相互作用的金属螯合纯化系统相比,NTA 与镍的结合更紧密。额外的螯合位点可防止镍离子浸出,与其他金属螯合纯化系统相比,它的结合能力更强,制备的蛋白纯度更高。QIAexpress 系统可用于从任何表达系统中纯化 His 标签蛋白,包括杆状病毒、哺乳动物细胞、酵母和细菌。

Procedure

His 标签蛋白的纯化分为 4 个阶段:细胞裂解、结合、洗涤和洗脱(请参阅 使用 Ni-NTA 蛋白纯化系统进行蛋白纯化)。使用 QIAexpress 系统纯化重组蛋白与蛋白或 6xHis 标签的三维结构无关。这样就可以在非变性或变性条件下,从稀释溶液和粗裂解物中一步纯化蛋白。强变性剂和洗涤剂可用于高效溶解和纯化受体、膜蛋白和形成包涵体的蛋白。洗涤缓冲液中可加入能高效去除非特异性结合污染物的试剂(见表)。通过添加 100–250 mM 咪唑作为竞争剂或降低 pH 值,在温和条件下洗脱纯化的蛋白。

与 His/Ni-NTA 相互作用兼容的试剂
变性剂洗涤剂还原剂其他用于长期储存
6 M Gu·HCl2% Triton X-10020 mM β-ME50% 甘油4 M MgCl2高达 30% 的乙醇
8 M 尿素2% 吐温 2010 mM DTT20% 乙醇5 mM CaCl2或 100 mM NaOH
1% CHAPS20 mM TCEP20 mM 咪唑2 M NaCl
See figures

Applications

Ni-NTA 基质能可靠地一步纯化适用于以下任何应用的 His 标签蛋白:

  • 结构和功能研究
  • 结晶以确定三维结构
  • 蛋白间和蛋白与 DNA 之间相互作用检测
  • 通过免疫接种产生抗体
  • 将纯化规模扩大到生产规模

Supporting data and figures

Specifications

FeaturesSpecifications
Applications蛋白质组学
Tag6xHis 标签
Bead size45–165 µm
Special feature批量和柱纯化
FPLC
Processing手动
Support/matrixSepharose CL-6B
Start material细胞裂解物
Scale大规模
Binding capacity最多 50 mg/ml
Gravity flow or spin column重力流
Yield100 µg – 100 mg

Resources

安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
试剂盒操作手册 (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