Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glutathione is somehow modifying/inhibiting my protein in some way, despite having removed the glutathione from the buffer via dialysis/ion exchange. I don't see anything out of the ordinary in my electron density that would suggest that glutathione has affected my protein in some way, but the huge difference seen in my activity assay suggests otherwise.
My question is, has anyone else seen an effect from glutathione affecting their protein in some way? My second question is, what's the minimum amount of glutathione necessary to elute your protein from a column? Sorry for the off topic question and thanks for any responses, Peter
