These are the usual culprits My buffers for cleavage and an on-column digestion worked good. (see below)
Also most likely your TEV source (do not go cheap) enzyme is inactive (gone bad). Get a clone for TEV and make your own TEV in the lab. It save you a ton of money . 10 mM Tris-HCl (pH 8.0) 150 mM NaCl 0.1% IGEPAL CA-630 0.5 mM EDTA 1 mM DTT (add immediately before use from 1 M stock) Best wishes P On Sat, May 2, 2015 at 10:56 AM, Giulliana Rangel < giulliana.ran...@gmail.com> wrote: > 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 > -- P
