产品信息

• 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

• 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

• 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）

• 注意事项

  1. 可在此处找到包含操作说明的详细产品手册。
  2. 若要长期贮存（> 30 天）E. coli ER2523（NEBExpress），推荐贮存于 -80℃。
  3. 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。
  4. MBP6-TEV-副肌球蛋白-ΔSal 是 TEV 蛋白酶切割的阳性对照。

• 参考文献

  1. Kapust and Waugh (1999). Protein Science. 8, 1668-1674.
  2. Guan, C. et al. (1987). Gene. 67, 21–30.
  3. Maina, C.V. et al. (1988). Gene. 74, 365–373.
  4. Amann, E. et al. (1985). Gene. 40, 183–190.
  5. Duplay, P. et al. (1984).  Biol. Chem.. 259, 10606–10613.
  6. Kellerman, O.K. et al. (1982). Methods in Enzymology. 90, 459–463.

操作说明、说明书 & 用法

• 操作说明

  1. NEBExpress MBP Fusion and Purification System Quick Start Protocol (NEB #E8201)
  2. Cloning a PCR Fragment Into a pMAL Expression Vector (E8201)
  3. Transformation Protocol (NEB #E8201)
  4. Small Scale Affinity Chromatography (NEB #E8201)
  5. Large Scale Affinity Chromatography (NEB #E8201)
  6. Cleavage of the Fusion Protein
  7. Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201)
  8. NEBuilder Assembly of a PCR Fragment

• 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

• 选择指南

  • Protein Expression and Purification Selection Chart

FAQs & 问题解决指南

• FAQs

  1. What strain(s) do you recommend as hosts for the pMAL vectors?
  2. What primers should I use to sequence the ends of my insert after I clone it into a pMAL vector?
  3. 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?
  4. How many times can I use the amylose column?
  5. What is known about binding in the presence of nonionic detergents?
  6. Can I substitute a different buffer and/or salt concentration in the Column Buffer?
  7. Can I perform a batch purification using the amylose resin?
  8. Can MBP fusions be purified in the presence of denaturants like urea or guanidine-HCl?
  9. How can TEV Protease be removed from the reaction after cleavage?
  10. Is the rate of TEV Protease cleavage affected by urea, guanidine hydrochloride and/or SDS?
  11. How do I separate MBP and TEV Protease from the protein of interest?
  12. I want to rebind MBP to the amylose column, but the maltose must be removed. Can this be done by dialysis?
  13. How should I store my protein after it is purified?
  14. What is MBP6? Is it different from wild-type MBP produced from E. coli?
  15. What gravity column do you recommend?
  16. Is the amylose resin damaged by storage at -20°C?

• 问题解决指南

  • Troubleshooting Guide for NEBExpress® MBP Fusion and Purification System (NEB #E8201)