Ni-NTA Superflow 96 BioRobot Kit

用于 6xHis 标记蛋白的中型自动纯化

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Ni-NTA Superflow 96 BioRobot Kit (4)

Cat. No. / ID: 969261

用于 4 x 96 6xHis 标记的蛋白制剂：4 个 QIAfilter 96 孔板，4 个 TurboFilter 96 孔板，1 个 100 ml Ni-NTA Superflow

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Ni-NTA Superflow 96 BioRobot 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。

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✓ Knowledgeable and professional Product & Technical Support

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Features

全自动纯化，每孔高达 2 mg 蛋白
无交叉污染的高纯度蛋白
原生或变性条件下的纯化

Product Details

Ni-NTA Superflow 96 BioRobot Kit 可同时从 96 个样本中自动纯化多达 300 µg 的重组蛋白（标准方案，每个样本 5 ml 培养液）。通过 BioRobot Workstation，澄清的细菌细胞裂解物流到预装有 Ni-NTA Superflow 的过滤板上。这种独特的 96 孔金属螯合亲和层析模块可选择性地紧密结合 His 标记蛋白。提供了在原生或变性条件下纯化的即用型方案。

Performance

为了满足客户通过自动化程序获得更大量蛋白的需求，QIAGEN 对 Ni-NTA Superflow 96 BioRobot 方案进行了调整，使其能够处理更大量的培养物，以及纯化每孔高达 2 mg 的 His 标记蛋白。将程序中使用的 Ni-NTA Superflow 树脂量增加到每孔 200 µl，并优化了裂解缓冲液配方，可处理最多 25 ml（原生条件下纯化）或 15 ml（变性条件下纯化）培养物（高产方案）。细胞培养物转移到 24 孔区块中进行处理。生物量的相应增加可提高每孔纯 His 标记蛋白的产量（见表和图每孔纯 His 标记蛋白的毫克含量），从而可对同一批蛋白进行多项检测，并减少所需的蛋白制备次数，另见图自动化表达克隆筛选。一步纯化可在很大的产量范围内提供高纯度蛋白，并可纯化大型蛋白复合物。

使用改良版 Ni-NTA Superflow 96 BioRobot 方案后的 His 标记蛋白产量
His 标记蛋白	每孔总产量 (µg)*	蛋白浓度 (mg/ml)†
绿色荧光蛋白	4000	6.0
T7 RNA 聚合酶	1000	1.4
GroES	300	0.4
氯霉素乙酰转移酶	2400	4.4
GroEL	740	1.0
肿瘤坏死因子 α	1600	2.5
GroES/GroEL	1200	1.5
Cpn-10	170	0.3

* 在两个 550 µl 洗脱馏分中获得的产量（6 次独立纯化的平均值）。80% 的蛋白在第一个 550 µl 中洗脱。
† 第一个 550 µl 洗脱馏分中的蛋白浓度。

See figures

每孔纯 His 标签蛋白的毫克含量。

自动化表达克隆筛选。

Principle

QIAexpress Ni-NTA 蛋白纯化系统，包括 Ni-NTA Superflow 96 BioRobot 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。（见图使用 Ni-NTA 蛋白纯化系统进行蛋白纯化）。

See figures

Procedure

His 标记蛋白的纯化分为 4 个阶段：细胞裂解、结合、洗涤和洗脱，是 Ni-NTA Superflow BioRobot Kit 的基础（见图）。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。

Ni-NTA Superflow 96 BioRobot Kit 适用于 BioRobot 3000、8000 或 9600 工作站。。

与 His/Ni-NTA 相互作用兼容的试剂
变性剂	洗涤剂	还原剂	其他	盐	用于长期储存
6 M Gu·HCl	2% Triton X-100	20 mM β-ME	50% 甘油	4 M MgCl₂	高达 30% 的乙醇
8 M 尿素	2% 吐温 20	10 mM DTT	20% 乙醇	5 mM CaCl₂	或 100 mM NaOH
	1% CHAPS	20 mM TCEP	20 mM 咪唑	2 M NaCl

See figures

Ni-NTA Superflow 96 BioRobot Kit。

Applications

QIAexpress Ni-NTA 蛋白纯化系统可为以下多项应用提供可靠的一步蛋白纯化：

结构和功能研究
结晶以确定三维结构
涉及蛋白间和蛋白与 DNA 之间相互作用的检测
通过免疫接种产生抗体

Supporting data and figures

自动化表达克隆筛选。

用于表达克隆筛选和蛋白表征的中型纯化。将用于表达 12、15 和 28 kDa 蛋白的载体构建体和 48/50 kDa 异二聚体的混合物转化到大肠杆菌中，接种在选择性培养基上，并随机挑选了 96 个菌群接种 5 ml 培养物。用 IPTG 过夜诱导了 6xHis 标签蛋白的表达。对细胞进行了造粒和裂解，并纯化了 6xHis 标签蛋白。每个洗脱馏分取 5 µl 用于 SDS-PAGE 分析；通过考马斯染色法对蛋白进行了显色。

Resources

Download Safety Data Sheets for QIAGEN product components.

For Automated medium and large-scale purification of 6xHis-tagged proteins

The following protocols have been designed for the use of Ni-NTA Superflow Columns on the QIAvac 6S vacuum manifold or in gravity-flow applications on the QIArack. Up to 15 mg 6xHistagged protein can be purified per column from cleared lysate derived from up to 1 liter of (E. coli) bacterial culture.

The two protocols given below are for the use of the Ni-NTA Superflow 96 BioRobot® Kit in manual procedures. The kit has been specially designed and optimized for automated 6xHis-tagged protein purification on QIAGEN® BioRobot Systems. For more details of the advantages of BioRobot Systems see the Ni-NTA Superflow 96 BioRobot Kit Handbook supplied with the kit or contact one of the QIAGEN Technical Service Departments or local distributors listed on the last page of the handbook.

For Automated medium and large-scale purification of 6xHis-tagged proteins

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

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

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

Yes, please follow either of the QIAGEN Supplementary Protocols:

FAQ ID -967