Thrombin cleaves Arg-Gly.

1. Wash GST-fusion protein bound on glutathione beads 3 times with the thrombin cleavage buffer (20 mM Tris-HCl pH8.5, 100 mM NaCl, 0.3 mM CaCl₂, 1 mM DTT, and 0.1% Tween X-100).

2. Add thrombin: ~1 unit/mg protein in 2 volumes of the thrombin cleavage buffer (i.e., 2 ml buffer for 1 ml of beads). The amount of thrombin needed may be determined by a small scale trail.

3. Incubate at room temperature or 4 °C for 2 hours to overnight.

4. Verify the cleavage is complete by SDS-PAGE.

5. Add thrombin if the cleavage is not complete and continue incubation.

6. Centrifuge to pack the beads. Transfer supernatant to a fresh tube. Protein will be in the flow through if glutathione beads were packed in a column.

7. Regenerating the column by eluting GST.

8. Thrombin can be separated from the sample protein by gel filtration. Alternatively, one may use the biotin-thrombin for cleavage and then streptavidin-agarose to remove biotin-thrombin.