Hi Greg, I am not sure why you are so surprised! If the zinc is altering the conformation and/or folding of your protein, this might change the accessibility of trypsin cleavage sites, thus changing your limited proteolysis pattern.
Eg: Metal-ion induced conformational changes in alkaline phosphatase from E. coli assessed by limited proteolysis V. Bučević-Popovića, M. Pavela-Vrančiča, , and R. Dieckmannb Biochimie Volume 86, Issue 6, June 2004, Pages 403-409 http://bit.ly/h2EtHj HTH, Dave ============================ David C. Briggs PhD Father, Structural Biologist and Sceptic ============================ University of Manchester E-mail: david.c.bri...@manchester.ac.uk ============================ http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB ============================ On 16 March 2011 09:52, Greg Carter <greg.carter...@gmail.com> wrote: > 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 >> **************************************************** >
