Dear all, Sorry for the non-crystallographic question. Currently I am working on a zinc binding protein which is expressed in insect cells and may contain 4-6 zinc ions. As we know, so many zinc binding proteins can absorb the iron ions from the culture medium and the protein looks from yellow to dark red when concentrated. But when I concentrate the protein, I didn’t see the red color even in the very high concentration. I am just wondering if a zinc binding protein is expressed from insect or mammalian cells, can the zinc binding sites grab the irons instead of zinc or the zinc binding site can be empty loaded if there is not enough zinc in the culture medium? If so, do I need to include some zinc salt into the culture medium when doing expression or I can add some zinc ions when purifying? Usually, how much zinc and at which step of purification can we add the zinc into the solution when doing purification?
Another question is that we know DTT can react with the heavy atoms to form the insoluble sulfide precipitates and if the zinc binding protein is purified with DTT at a final concentration of 1-5 mM, can it strip the zinc ions from the protein? I am appreciated if someone has this kind of experimental experiences and thanks in advance! Heng
