Have you analysed the cleavage by SDS-PAGE?
You should run samples of the slurry before and after cleavage to see if the
thrombin has cleaved your protein. (~2-5uL of the 50% slurry should be
enough/lane)
A few suggestions if there is no apparent cleavage.
(1) digest up to 3 h at 37degC (assuming your protein is relatively stable)
(2) prior to cleavage, add 5 mM DTT to 50% slurry of beads with GST-fused
protein bound. Incubate for 1 h at room temp then wash out DTT with at least
5 column volumes of thrombin cleavage buffer. Then add thrombin to digest
(preferably at 37 degC).
Good luck.
John.
"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
>
