His tag cleavage by Thrombin - (Feb/17/2006 )

I've tried to cleave His-tag with Thrombin. But there are several problems.

1. The protein is not soluble at neutral pH , but at pH 9.5.
When I treated thrombin at this pH, I found many bands that looked like the products of nonspecific digestion. But there were also a lot of intact his-tagged protein.

I just wonder whether thrombin is working specifically at pH 9.5.

2. Also what is better to treat thrombin on the column or to the eluated protein?

Thank you for your replies

you may check your protein sequence carefully. Thrombin can cut following peptides:

P4-P3-Pro-Arg/Lys-P1-P2, where P3 and P4 are hydrophobic amino acids and P1 and P2 are nonacidic amino acids.

P2-Arg/Lys-P1, where P2 or P1 are Glycine.

I think most likely your protein meets the second condition somewhere.

Good luck