I'm working on a protein with a GST tag followed by a His tag. To remove the GST tag, I performed thrombin digestion at 4°C in the buffer containing 500 mM NaCl, 50 mM Tris (pH 8), and 5 mM β-mercaptoethanol. At the 12-hour mark, cleavage was only partial, so I extended the duration of the digestion to 4 more hours (16 hours in total). Further, I performed NiNTA Re-affinity. The GST was eluted during elution, and my protein was detected in the flow-through and column wash. However, the total yield of the protein was very low.

Previously, in the buffer with only 20 mM Tris and 5 mM β-mercaptoethanol (no NaCl), complete digestion occurred at the 12th hour.

Could the high salt concentration (500 mM NaCl) have affected the thrombin activity? What other factors might have affected the cleavage?