Ni-NTA Fast Start Kit

用于纯化和检测来自大肠杆菌裂解物的 His 标记重组蛋白

✓ 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

用于纯化和检测六种 6xHis 标签蛋白：6 个快速启动纯化柱、Penta·His 抗体、缓冲液和试剂

Copy order details

Ni-NTA Fast Start Kit 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

蛋白质科学领域新手的理想选择
一应俱全的高效纯化试剂盒
易于理解的方案和简单明了的处理
可在短短 90 分钟内实现每个柱高达 25 mg 的蛋白产量

Product Details

Ni-NTA Fast Start Kit 提供了从澄清的大肠杆菌裂解物中快速、高效地纯化 His 标记蛋白所需的一切，包括预填充的 Ni-NTA 柱。试剂盒中提供的缓冲液可在原生或变性条件下纯化蛋白。试剂盒还含有一种抗 His 抗体，用于检测表达的 His 标记蛋白。

Performance

Ni-NTA Fast Start Kit 提供了从澄清的大肠杆菌裂解物中快速、高效地纯化 His 标记蛋白所需的一切。试剂盒中提供的缓冲液可在原生或变性条件下纯化蛋白（见图）简单明了的方案和即用型试剂使 Ni-NTA Fast Start Kit 成为希望将研究领域扩展到蛋白表达领域、但又不熟悉蛋白纯化程序的科学家的理想起点。

See figures

非变性或变性条件下的高纯度蛋白。

Principle

Ni-NTA Fast Start Kit 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。该技术可在原生或变性条件下，一步纯化几乎所有 His 标记蛋白。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，制备的蛋白纯度更高。His 标记还构成了可被试剂盒附带的 Penta His Antibody 识别的表位。

Procedure

细胞在提供的裂解缓冲液中进行裂解，然后经离心分离，得到澄清的裂解物。将该裂解物应用在 Fast Start Column 上，His 标记蛋白与柱实现高亲和力结合，而无标记蛋白则通过柱。洗涤后，使用含咪唑（原生条件）或低 pH 值（变性条件）的缓冲液洗脱纯化的蛋白（见图）。可通过 SDS-PAGE、蛋白印迹和使用提供的 Penta His Antibody 进行免疫检测来跟踪纯化程序和蛋白产量。

See figures

高纯度 His 标签蛋白。

Applications

Ni-NTA Fast Start Kit 能可靠地一步纯化适用于以下任何应用的 6xHis 标记蛋白：

功能研究
通过免疫接种产生抗体
涉及蛋白间和蛋白与 DNA 之间相互作用的检测
结构研究

Supporting data and figures

高纯度 His 标签蛋白。

Specifications

Features	Specifications
Applications	蛋白质组学
Support/matrix	Ni-NTA 基质
Processing	手动
Binding capacity	5–20 mg/ml
Special feature	Ni-NTA 技术
Gravity flow or spin column	重力流
Start material	细胞裂解物
Tag	6xHis 标签
Yield	每柱 <10 mg 蛋白

Resources

Download Safety Data Sheets for QIAGEN product components.

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

The Fast Start Columns in the QIAexpress Ni-NTA Fast Start Kit are prepacked with Ni-NTA Superflow resin.

FAQ ID -836

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

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

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

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

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