Giulliana,
Alternatively to the previous excellent suggestions, you can add a few more residues between your protein and TEV-cleavage site. For example, if you have tag on the C-terminus you can add GSGS after the last residue: it should improve cleavage without significant impact on crystallization. Vitali ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Giulliana Rangel <giulliana.ran...@gmail.com> Sent: Saturday, May 2, 2015 12:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
