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