Purifying yeast proteins using six his tag.
dfried at darwin.genetics.washington.edu
Fri Mar 24 17:56:19 EST 1995
In article <Pine.3.05.9503231821.A7896-9100000 at orchid.UCSC.EDU> sherif at ORCHID.UCSC.EDU (Sherif Abouelela) writes:
>Hi to all the yeast lovers:
> Does anybody know of a good way to avoid non specific binding of
>proteins to the Ni-NTA column when purifying yeast protein containing a
>six his tag. I can see my protein using immunoblotting, but lots of stuff
>appear when I stain. Any ideas or tricks
You should try to elute your protein from the column using a gradient of
either increasing imidazole or decreasing pH. Imidazole will compete with
your 6HIS tag for binding to the Ni-matrix, so contaminants that bind
better or worse than your 6HIS protein will elute differently. A 6HIS tag
should elute around 100 mM imidazole, according to the folks at Qiagen. A pH
gradient will work for similar reasons, but this time the lower pH will add
protons to the histidines and make them unable to associate with the
Ni-matrix. The lower the pH, the less able a given protein will be able to
bind to the matrix.
Hope this helps-
Dept. of Biochemistry, SJ-70 "I am not a number,
U of Washington, Seattle WA 98195 I am a Friedman!"
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