产品信息

• 产品组分信息

  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
  P8112S     -20       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

• 特性和用法

  单位定义

  1 unit of TEV Protease will cleave 2 µg of MBP-fusion protein, MBP5-TEV-paramyosin ΔSal, to 95% completion in a total reaction volume of 10 µl in 1 hour at 30°C in 50 mM Tris-HCl (pH 7.5 @ 25°C) with 0.5 mM EDTA and 1 mM DTT.

  反应条件

  1X TEV Protease Reaction Buffer
  Incubate at 30°C

  1X TEV Protease Reaction Buffer
  50 mM Tris-HCl
  0.5 mM EDTA
  1 mM DTT
  (pH 7.5 @ 25°C)

  贮存溶液

  50 mM Tris-HCl
  250 mM NaCl
  1 mM TCEP
  1 mM EDTA
  50% Glycerol
  pH 7.5 @ 25°C

  热失活

  65°C for 10 minutes

  分子量

  实际: 28 kDa

  单位活性检测条件

  Two fold dilutions of TEV Protease are incubated with 2 μg MBP5-TEV-paramyosin ΔSal and 1X TEV Protease Reaction Buffer in a 10 µl reaction. The reaction mix is incubated at 30°C for 1 hour. Separation of reaction products are visualized by SDS-PAGE.

• 优势和特性

  Features

  • Removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins
  • Engineered to improve thermal stability and decrease autolysis
  • High substrate specificity with no non-specific proteolysis 

   

• 注意事项

  1. The TEV Protease recognition sequence with the highest catalytic efficiency is ENLYFQ ▼S; however, the amino acid in the P1’ position (S in the recognition sequence) can also be G, A, M, C, or H (1).
  2. If the fusion protein sample contains > 2 M urea, > 0.5 M Guanidine hydrochloride, > 50 mM imidazole, pH values below 6 or above 9, or cysteine protease inhibitors then it will be necessary to dialyze the fusion protein before TEV Protease cleavage.
  3. TEV protease can be used at high concentrations without the occurrence of non-specific proteolysis.
  4. Reactions may be scaled-up linearly to accommodate larger reaction volumes.

• 参考文献

  1. Kapust, R.B. et al. (2002). Biochem. and Biophysical Research Comm.. 294, 949-955.

操作说明、说明书 & 用法

• 操作说明

  1. Typical Reaction Conditions for TEV Protease (NEB #P8112)
  2. His-tag removal from protein using TEV Protease

工具 & 资源

• 选择指南

  • Protease Selection Chart

FAQs & 问题解决指南

• FAQs

  1. Can TEV Protease recognize and cleave a sequence other than Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)?
  2. Is it necessary to dialyze the sample prior to cleavage with TEV Protease?
  3. Can TEV Protease be used at lower temperatures?
  4. Is TEV Protease compatible with protease inhibitors?
  5. Is TEV Protease compatible with different reaction buffers?
  6. Can TEV Protease be removed from the reaction after cleavage?

• 实验技巧

  • The optimal pH range is 6.0 – 8.0
  • Optimal activity achieved in ≤0.2M NaCl; however, the enzyme retains some activity in up to 2M NaCl

  Cleavage at lower temperatures, such as 4ºC, requires overnight incubation

  Inhibition occurs in the presence of ≥ 5 mM Zn²⁺, ≥ 1 mM Cu²⁺ and ≥ 10 mM Co²⁺

  • Compatible with 10mM MgSO₄, MnCl₂ and CaCl₂ and up to 100mM EDTA
  • Compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF
  • Can be used at high concentrations with no non-specific proteolysis occurring

  • Some substrates may require extended incubation (up to three days at either 4°C or 30°C)
  • If cleavage is not complete, add more TEV protease after 24 hours and continue incubation