上海金畔生物科技有限公司代理New England Biolabs(NEB)酶试剂全线产品,欢迎访问官网了解更多产品信息和订购。
产品信息
通过直链淀粉纯化后,便可使用 TEV 蛋白酶从 MBP 标签上将目的蛋白切割下来,不会在蛋白中添加任何源自载体的残基。MBP 标签和 TEV 蛋白酶均 His 标签,便于从反应中轻松去除。将前述反应产物经 NEBExpress 镍柱填料(NEB #S1428)分离,可去除 MBP 标签和 TEV 蛋白酶,从而在离心柱流经液中提取目的蛋白。目的蛋白的产量最高可达 100 mg/L,常规产量范围为 10 – 40 mg/L。
- 产品类别:
- Amylose Purification (MBP-tag),
- NEBExpress MBP Fusion and Purification System,
- Non-T7 Expression,
- Bacterial E. coli Protein Expression Products,
- Affinity Purification Products,
- Protein Purification Products,
- Protein Expression Products,
E. coli Expression Strains Products
- 应用:
- Target Protein Insolubility ,
- Non-T7 Expression,
- Expression of Difficult Proteins,
- Protein Expression in E. Coli,
Protein Expression
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试剂盒组成
本产品提供以下试剂或组分:
NEB # 名称 组分货号 储存温度 数量 浓度 -
E8201S Multi-temperature NEBExpress® MBP Fusion and Purification System E8201-1 -20 pMAL-c6T Vector N0378SVIAL -20 1 x 10 µg 200 µg/ml MBP6 Protein E1020AVIAL -20 1 x 10 µg 40 µg/ml MBP6-TEV-Paramyosin ΔSal E1021AVIAL -20 1 x 100 µg 5 mg/ml TEV Protease P8112SVIAL -20 1 x 0.1 ml 10,000 units/ml TEV Protease Reaction Buffer B8035SVIAL -20 1 x 1 ml 10 X Anti-MBP Monoclonal Antibody E8032AVIAL -20 1 x 0.025 ml Not Applicable E. coli ER2523 (NEB Express) (Glycerol Stock) E4131SVIAL -20 1 x 0.2 ml Not Applicable Amylose Resin E8021S 4 Amylose Resin E8021SVIAL 4 1 x 15 ml Not Applicable
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优势和特性
Features
- 可信赖的大肠杆菌表达:产量极高(高达 100 mg/L)
- 与 MBP 融合可显著促进目的蛋白的正确折叠
- 两步纯化:直链淀粉洗脱后进行 TEV 蛋白酶切割,再经镍柱填料分离获得高纯度的无标签目的蛋白
- 使用麦芽糖进行温和洗脱;无需去污剂或刺激性强的变性剂
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相关产品
相关产品
- Amylose Resin
- TEV Protease
- Anti-MBP Monoclonal Antibody
- AlwNI
- SbfI-HF®
- Monarch® PCR & DNA 纯化试剂盒(5 μg)
- Monarch® DNA 胶回收试剂盒
- 瞬时粘性末端连接酶预混液
- NEBuilder® 高保真 DNA 组装预混液
- 蓝色蛋白上样染料
- Unstained Protein Standard, Broad Range (10-200 kDa)
- Amylose Resin High Flow
- Amylose 磁珠
- 抗 MBP 磁珠
- NEBExpress® Ni-NTA 磁珠
- NEBExpress® Ni Spin Columns
- NEBExpress® Ni Resin
- c2523-neb-express-competent-e-coli-high-efficiency
- NEB® 10-beta E. coli 感受态细胞(高效级)
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注意事项
- 可在此处找到包含操作说明的详细产品手册。
- 若要长期贮存(> 30 天)E. coli ER2523(NEBExpress),推荐贮存于 -80℃。
- Amylose 树脂应贮存于 4℃,以防冻坏。不过,树脂性能不会因一次冻融循环而下降。
- MBP6-TEV-副肌球蛋白-ΔSal 是 TEV 蛋白酶切割的阳性对照。
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参考文献
- Kapust and Waugh (1999). Protein Science. 8, 1668-1674.
- Guan, C. et al. (1987). Gene. 67, 21–30.
- Maina, C.V. et al. (1988). Gene. 74, 365–373.
- Amann, E. et al. (1985). Gene. 40, 183–190.
- Duplay, P. et al. (1984). Biol. Chem.. 259, 10606–10613.
- Kellerman, O.K. et al. (1982). Methods in Enzymology. 90, 459–463.
操作说明、说明书 & 用法
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操作说明
- NEBExpress MBP Fusion and Purification System Quick Start Protocol (NEB #E8201)
- Cloning a PCR Fragment Into a pMAL Expression Vector (E8201)
- Transformation Protocol (NEB #E8201)
- Small Scale Affinity Chromatography (NEB #E8201)
- Large Scale Affinity Chromatography (NEB #E8201)
- Cleavage of the Fusion Protein
- Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201)
- NEBuilder Assembly of a PCR Fragment
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说明书
产品说明书包含产品使用的详细信息、产品配方和质控分析。- manualE8201
工具 & 资源
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选择指南
- Protein Expression and Purification Selection Chart
FAQs & 问题解决指南
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FAQs
- What strain(s) do you recommend as hosts for the pMAL vectors?
- What primers should I use to sequence the ends of my insert after I clone it into a pMAL vector?
- What is the minimum size of a fragment that can be cloned into pMAL and expressed fused to MBP? Can short peptide sequences (~ 10 amino acids) be added onto MBP?
- How many times can I use the amylose column?
- What is known about binding in the presence of nonionic detergents?
- Can I substitute a different buffer and/or salt concentration in the Column Buffer?
- Can I perform a batch purification using the amylose resin?
- Can MBP fusions be purified in the presence of denaturants like urea or guanidine-HCl?
- How can TEV Protease be removed from the reaction after cleavage?
- Is the rate of TEV Protease cleavage affected by urea, guanidine hydrochloride and/or SDS?
- How do I separate MBP and TEV Protease from the protein of interest?
- I want to rebind MBP to the amylose column, but the maltose must be removed. Can this be done by dialysis?
- How should I store my protein after it is purified?
- What is MBP6? Is it different from wild-type MBP produced from E. coli?
- What gravity column do you recommend?
- Is the amylose resin damaged by storage at -20°C?
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问题解决指南
- Troubleshooting Guide for NEBExpress® MBP Fusion and Purification System (NEB #E8201)