Ni-NTA Magnetic Agarose Beads

用于高通量、微型 His 标记蛋白纯化，以及使用 His 标记进行多功能磁性捕获检测

Products

Features

用于增强信噪比和重复性的定向呈现
可通过改变磁珠数量实现多种结合能力
即使使用粗细胞裂解物也能进行有效的筛选程序
研究生物分子相互作用的理想选择
全自动化选项

Product Details

Ni-NTA Magnetic Agarose Beads 是涂有 Ni-NTA Agarose 亲和纯化基质的磁微粒。它们用于固定和纯化带有 His 标记的重组蛋白。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。蛋白结合后，可使用磁铁将微珠沉淀、洗涤，然后在原生或变性条件下用少量缓冲液洗脱蛋白。

Principle

QIAexpress Ni-NTA 蛋白纯化系统，包括 Ni-NTA Magnetic Agarose Beads（见图 Ni-NTA Magnetic Agarose Beads 的显微图），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。该技术可在原生或变性条件下，从任何表达系统中一步纯化几乎所有 His 标记蛋白（见图使用 Ni-NTA 蛋白纯化系统进行蛋白纯化）。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。

See figures

Ni-NTA Magnetic Agarose Beads 的显微图。

微型蛋白纯化。

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（见图微型蛋白纯化）。使用 QIAexpress 系统纯化重组蛋白与蛋白或 His 标记的三维结构无关。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	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

微型蛋白纯化。

Applications

QIAexpress Ni-NTA 蛋白纯化系统（包括 Ni-NTA Magnetic Agarose Beads）可为以下任何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

微型蛋白纯化。

Resources

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

Modulating RssB activity: IraP, a novel regulator of sigma(S) stability in Escherichia coli.

Bougdour A; Wickner S; Gottesman S;

Genes Dev; 2006; 20 (7):884-97 2006 Apr 1 PMID:16600914

Bending fatigue study of nickel-titanium Gates Glidden drills.

Luebke NH; Brantley WA; Alapati SB; Mitchell JC; Lausten LL; Daehn GS;

J Endod; 2005; 31 (7):523-5 2005 Jul PMID:15980713

Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay.

Day-Storms JJ; Niranjanakumari S; Fierke CA;

RNA; 2004; 10 (10):1595-608 2004 Aug 30 PMID:15337847

Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Suer S; Sickmann A; Meyer HE; Herberg FW; Heilmeyer LM Jr;

Eur J Biochem; 2001; 268 (7):2099-106 2001 Apr PMID:11277933

FAQ

When working with small amounts of 6xHis-tagged protein in dilute solution, such as proteins expressed in mammalian cells or secreted into cell-culture medium, we recommend using Ni-NTA Magnetic Agarose Beads.

The total binding capacity of Ni-NTA Magnetic Agarose Beads is 3 µg of protein per 10 ul of magnetic bead suspension. Adjusting the amount of beads to the amount of 6xHis-tagged protein to be captured is crucial for optimal performance. The small elution volumes used provide high 6xHis-tagged protein concentrations, and allow detection of the purified proteins using Coomassie-stained SDS polyacrylamide gels.

Please see protocol 15 in the QIAexpressionist Handbook for detailed descriptions of a procedure to purify 6xHis-tagged proteins from transfected mammalian cells.

FAQ ID -134

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

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 the Supplementary Protocol 'Purification of 6xHis-tagged proteins using the BioSprint 96 Workstation' (BS17).

FAQ ID -1167

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