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
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.