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Hey Giulliana, does your protein buffer contain high imidazole concentrations ( > 150 mM)? If so you should try to exchange the buffer before cleavage, since the TEV protease tends to precipitate at higher imidazole concentrations. Schara > Am 02.05.2015 um 19:56 schrieb Giulliana Rangel <[log in to unmask]>: > > 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