His-tag removal from protein using TEV Protease

TEV Protease (NEB# P8112) is recommended for cleavage of a His-tag following purification with NEBExpress™ Ni-NTA Magnetic Beads, Ni Spin Columns or Ni Resin. First, the expression vector must be designed to contain a TEV site between the His-tag and the protein. Optimal incubation times and enzyme concentrations must be determined empirically for a particular protein. Reactions may be scaled-up linearly to accommodate larger sample amounts and reaction volumes. Typical reaction conditions are as follows:

  1. Dialyze the protein against 20 mM Tris-HCl, pH 7.5

  2. Determine the protein concentration.

  3. Combine 15 μg of protein and H2O (if necessary) to make a 45 μl total reaction volume.

  4. Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.

  5. Add 1 μl of TEV Protease.

  6. Incubate at 30°C for 1 hour or at 4°C overnight.