Typical Reaction Conditions for TEV Protease (NEB #P8112)
Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Reactions may be scaled-up linearly to accommodate larger sample amounts and reaction volumes. Typical reaction conditions are as follows:
- Combine 15 μg of substrate and H2O (if necessary) to make a 45 μl total reaction volume.
- Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.
- Add 1 μl of TEV Protease.
- Incubate at 30°C for 1 hour or at 4°C overnight.
- 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 into TEV protease reaction buffer before TEV Protease cleavage.
- TEV protease is inhibited by reaction buffers containing >40% Glycerol.
- 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.
- Optimal activity achieved in ≤0.2M NaCl; however, the enzyme retains some activity in up to 2M NaCl.
- Some substrates may require extended incubation periods (up to three days at either 4°C or 30°C) to achieve complete cleavage. The addition of more TEV Protease after 24 hours may also help achieve complete cleavage of some substrates.