Recombinant Tobacco Etch Virus Protease (rTEV)

1. TEV Protease;
2. 10X TEV Protease Buffer + Salt: 500mM Tris-HCl, pH8.0, 500mM NaCl, 5mM EDTA, 10mM DTT;
3. 10X TEV Protease Buffer – Salt: 500mM Tris-HCl, pH8.0, 5mM EDTA, 10mM DTT.

1. Add the following to a microcentrifuge tube:Add the following to a microcentrifuge tube:

2. Mix and incubate at 30°C, Remove 10 μL aliquots at 1, 2, 4, and 6 hours.

3. Analyze by SDS-PAGE.

Guidelines for Cleavage

• For most fusion proteins, TEV Protease functions optimally in a reaction mixture without NaCl; however, conditions may be optimized by varying the NaCl concentration from 0 mM to 500 mM. Remember to take into account the contribution of salt from the enzyme and from your substrate. When setting up your cleavage reaction, use the appropriate 10X rTEV Protease Buffer +/- Salt.
• Researchers need to optimize their specific reaction conditions. As an initial suggestion, 10 units of rTEV protease can be used per 30μg of target protein for 1 hour at 30 °C, or overnight at 2–8 °C. The cleavage efficiency can then be estimated by SDS-PAGE.