Histag protein purification problem
Dr E. Buxbaum
EB15 at le.ac.uk
Tue Apr 1 04:32:44 EST 1997
"Dr. Thomas T. chen" <chen at uconn.edu> wrote:
>I used pET system to express histag fused peptide in BL21 (DE3). When I
>purified the fusion peptide by MCAC, I found other bacterial proteins
>sticking to the column tightly. I tried to use thrombin to cut the fusion
>peptide in order to purify the target peptide, but without success. Can
>anyone give me suggestion how to purify my target peptide?
> Thank in advance.
In my experience the one-step affinity purification of tagged proteins is
a dream that often remains unfulfilled. Use the affinity column as a
first purification step, to get rid of most of the rubbish and to
concentrate your protein. With the proteins eluted from the column then
do some other purification like IEC, HIC, gelchromatography. As the
affinity chromatography has already done most of the work, a single
further step, if carefully worked out, will be sufficient in most cases.
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