Unexplained His-tag cleavage - (Dec/15/2005 )
Hello everyone, I have a question about some his-tagged protein I've been working with lately. We recently started monitoring our cleavages via HPLC, and while we are not 100% sure, it seems that some of our his-tagged protein is becoming cleaved before we digest our protein (with thrombin). Has anyone ever seen this happen before? His-tags seemingly becoming cleaved without any proteases being presented. Maybe possible that there is some other protease present there that we do not know about? Any help would be appreciated, thanks.
have you tried running the amino acid sequence on a peptide cutter program? if not, try here: http://us.expasy.org/tools/peptidecutter/
if you are purifying from crude cell lysates it is quite possible that protases are chewing up your protein. there are proteases that could also be co-eluting with your protein, undetectable with coomasie stain. try a protease inhibitor and see if it stops/slows it down. i use Roche Complete with no EDTA.
hope that helps!
if you are purifying from crude cell lysates it is quite possible that protases are chewing up your protein. there are proteases that could also be co-eluting with your protein, undetectable with coomasie stain. try a protease inhibitor and see if it stops/slows it down. i use Roche Complete with no EDTA.
hope that helps!
I'll take a look at that peptide cutter program, looks pretty nifty. We usually do use PMSF in our sonnication steps, however we did leave it out in this prep. and we run our sonnicate right onto our Ni column. Thanks for your help.