GSH will reduce your protein quite nicely - is your enzyme activity redox sensitive?
--- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: [email protected] tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 8/29/12 5:56 PM, "Peter Hsu" <[email protected]> wrote: >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
