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Peter, Such high concentration of GSH may change the pH according to our experience. We usually use 50 mM Tris pH=8.0, 5 mM GSH, 3 mM DTT - so that you can load the sample on ion-exchange column after elution. If your protein is not stable without NaCl - you should add it also. Vitali On Wed, Aug 29, 2012 at 12:42 PM, Peter Hsu <[log in to unmask]>wrote: > I don't think so since I purify in the presence of reducing agents > (DTT/BME) and I got activity out of the prep that was released by on column > cleavage. On the other hand, I don't usually add those fresh each time I > use the buffers so it's entirely possible the reducing agents are nowhere > near the initial reduced form they were in initially. > > > On Wed, 29 Aug 2012, Antony Oliver wrote: > > 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: [log in to unmask] >> tel (office): +44 (0)1273 678349 >> tel (lab): +44 (0)1273 677512 >> >> >> >> >> >> >> On 8/29/12 5:56 PM, "Peter Hsu" <[log in to unmask]> 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 >>> >> >> >>