Hi Matthew,
    TEV protease is very robust. I normally digest with 1:100 ratio according to the OD280. I normally digest at 4C for overnight around 16-18 hours. Make sure your tev protease site are not inaccessible and buried inside.


On Mon, May 24, 2010 at 12:27 PM, Matthew Merski <[log in to unmask]> wrote:
Hello all,

 I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0.  Has anyone been able to overcome a similar problem?

Matthew Merski
Post-doctoral researcher