To add more information: The proteolysis buffer was 50 mM Tris / HCl pH 8.0, 150 mM NaCl, 0.5 mM ZnCl and 0.1 mM TCEP; protein concentration was ~ 25 µM. Proteolysis was carried out at 4°C over 2 hours.
Thank you very much for the literature, Mark - I'll look into it. Greg 2011/3/16 Matthias Zebisch <matth...@strubi.ox.ac.uk> > how much zinc would be essential to know... > > > > Am 16/03/2011 09:18, schrieb Greg Carter: > > Dear all, > > I was working with a protein which is known to bind zinc. I tried to make a > limited proteolysis (with trypsin) after purification (metal affinity, ion > exchange and gel filtration; last step uses EDTA to remove bound metal ions) > in the presence and absence of zinc ions and I was quite surprised that the > proteolysis pattern is completely different although all parameters were the > same during the proteolysis (except for the presence of zinc ions). Since > the protein I'm using is His-tagged (and I did not remove the His-tag), I > was wondering whether anybody of you knows if zinc > > 1.) affects trypsin in it's activity? > 2.) zinc can bind to the His-tag and affects the result of the limited > proteolysis? > 3.) zinc does not have any effect on His-tagged proteins? > > Just another comment: I also tried the same proteolysis in the presence of > magnesium and manganese, but the proteolysis pattern looks the same as the > one without metal ions. > > Any comments are welcome, > > Greg > > > > -- > **************************************************** > Dr. Matthias Zebisch > The Division of Structural Biology > The Henry Wellcome Building for Genomic Medicine > Roosevelt Drive > Oxford, OX3 7BN > United Kingdom > Phone : +44-1865-278549 (office) > Mobile: +44-786-6841877 > Fax : +44-1865-2785 > email: matth...@strubi.ox.ac.uk > **************************************************** > >
