1) cutting on column often does not work. Cleavage in solution is generally
a safer alternative. Make sure that cleavage actually HAPPENS in solution
before going to column cleavage - you may discover that steric reasons
prevent efficient cleavage in your particular case.
2) proteins often stick to glutathione columns nonspecifically (use reducing
agent + detergent to try and wash them off)
3) try different protease sites (yes that means re-cloning, but that's only
a few days' worth of time these days) (your thrombin may have become
inactivated, there's several possible reasons for that)
4) use a different tag :)
A.G.E.
"Chan Shzu Wei" <mdccsw at nus.edu.sg> wrote in message
news:BCED9767330536479E416EFC52D3F9B9038E990B at pfs01.ex.nus.edu.sg...
> Dear colleagues,
>> I have problem in purifying my protein of interest from the GST from
> using GST column. I have cut my protein of interest (~25.7kDa) from the
> GST fusion protein (26 kDa) with thrombin protease, and as in theory, I
> should get my protein of interest in flow through, but it didn't. I
> found that it came out together with the GST when I eluted with the GST
> elution buffer. Please give some suggestion to purify my protein of
> interest from the GST protein. Thanks.
>> Joyce
>
