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
