快速、小规模纯化His-tagged蛋白,同时提供离心柱、试剂、缓冲液和对照质粒

  • 15分钟内纯化至多300 µg His-tagged蛋白
  • 可在天然和变性条件下纯化
  • 一步法即可纯化得到95%的产物
  • 即用型离心柱,可用于快速自动化或手工纯化

Ni-NTA硅胶基质通过大孔硅胶与Ni-NTA相结合来优化材料,从而抑制相互之间的非特异性疏水作用。Spin Kit中的Ni-NTA Columns为便捷的小离心柱形式,可便利地处理多份平行样本。适用于功能性筛选工程蛋白质、选择克隆表达性长片段转录产物,以及表达水平比较。每个离心管可以纯化至多300 µg的His-tagged蛋白。Ni-NTA离心管可以在天然或变性的条件下,一步法纯化蛋白质。Ni-NTA Spin Kit是离心纯化His-tagged蛋白的完整试剂盒。

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Ni-NTA Spin Kit (50)
50 Ni-NTA Spin Columns, Reagents, Buffers, Collection Tubes, 1 μg Control Expression Plasmid
31314
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The Ni-NTA Spin Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Ni-NTA Spin Column purification with the Ni-NTA.|Reproducible automated purification.|Purification at different expression levels.|
|The indicated proteins were purified in duplicate under native conditions using Ni-NTA Spin Columns from cleared E. coli cell lysates derived from 5 ml LB cultures either manually or in an automated procedure on the QIAcube. CAT: chloramphenicol acetyl transferase; GFP: Green fluorescent protein; HIV-RT: Human immunodeficiency virus reverse transcriptase; IL-1b: Interleukin-1 beta. M: markers; C: cleared lysate (2 μl loaded per lane); E: elution fraction (3 μl loaded per lane).



 |The 6xHis-tagged mouse DHFR was expressed at the indicated levels in E. coli, purified from 3 ml cultures using the Ni-NTA Spin Kit under denaturing conditions, and eluted in buffer at pH 5.9. Fractions were visualized by Coomassie staining after SDS-PAGE. 5 µl (of each 200 µl eluate) was loaded. 1: cell lysate; 2: flow-through; 3: first eluate; 4: second eluate.|

Performance
Ni-NTA Spin Kit是快速、高效纯化蛋白质的理想试剂(参见Purification at different expression levels),同时也呈现了高度的可重复性(参见Reproducible automated purification)。
Principle

QIAexpress Ni-NTA Protein Purification System,包括Ni-NTA Spin Columns,原理是基于著名专利技术Ni-NTA(镍-次氮基三乙酸)树脂,用于纯化带有6个或者以上组氨酸残基His标签的蛋白质。该技术能够在天然或者变性的条件下,从任何表达体系中,一步法纯化大多数His-tagged蛋白。NTA有4个镍离子的螯合位,相比金属-螯合纯化系统只有3个与金属离子相互作用的位点,能够更紧密地与镍结合。额外的螯合位点防止镍离子脱落,从而拥有更强的结合能力,并且相比于其他金属-螯合纯化系统,该系统能制备更高纯度的蛋白质。QIAexpress体系可以从任何的表达体系中,包括杆状病毒、哺乳动物细胞、酵母和细菌,纯化His-tagged蛋白。

Procedure

纯化His-tagged蛋白包括4个步骤:细胞裂解、蛋白结合、洗涤和洗脱(参见"Ni-NTA Spin Column purification with the Ni-NTA")。利用QIAexpress系统纯化重组蛋白并不取决于蛋白质或者His标签的三维空间结构。这一特性能够在天然或变性条件下,一步从稀释溶液或者粗裂解物中纯化蛋白。多至600 μl细胞裂解物可加注到Ni-NTA离心柱上。标签蛋白在2分钟快速离心结合到Ni-NTA硅胶基质上,无标签蛋白则流过基质。在洗涤步骤后,纯化的蛋白质在温和条件下洗脱(如pH降至5.9,或者加入100–500 mM咪唑)至100–300 μl体积。因为His标签较小,几乎不产生免疫原,通常无需去除。纯化的蛋白可以立即使用。蛋白质可以从多个小规模的表达培养物中纯化得到,只需约30分钟或者60分钟(自动化的QIAcube全自动核酸纯化仪操作流程)。强变性剂和清洁剂可以有效地溶解和纯化受体、膜蛋白和形成包涵体蛋白质。这些试剂能够高效地去除非特异性结合的污染物,可以包含在洗涤缓冲液中(参见下表)。在温和的条件下,加入100–250 mM咪唑作为竞争物或者降低pH值洗脱纯化的蛋白质。

与Ni-NTA–His兼容的试剂
试剂
6 M guanidine HCl
8 M尿酸
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
所列的试剂已成功地运用在所给的浓度上限中。.
Applications

QIAexpress Ni-NTA Protein Purification System提供可靠的一步法纯化蛋白质,适用于各种应用,包括:

  • 结构和功能研究
  • 蛋白结晶用于三维结构分析
  • 蛋白与蛋白,蛋白与DNA相互作用研究
  • 抗体制备
Feature
Specifications
Applications Proteomics
Bead size 16–24 µm
Binding capacity Up to 300 µg per spin column
Gravity flow or spin column Spin column
Processing Automated
Scale Small scale
Special feature Up to 95% homogeneity in one step
Start material Cell lysate
Support/matrix Macroporous silica
Tag 6xHis tag

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