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
>
>
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