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I think your problem is caused by a high protein concentration.
Since your protein is soluble before the His column (which concentrates the
protein).
Not necessarily. We've come accross several cases where E.coli
expressed (MBP fusions most frequently) are in *soluble* aggregates.
>90% of total protein then fails to bind to affinity columns and
the purified protein usually precipitates upon cleaving MBP off.
Try to find a stabilization buffer, add glycerol or change pH.
Another crucial parameter could be the redox environement. Which will be
difficult to test because you use a his tag. Change the tag to GST, you can
then use 10mM DTT in your buffer.
Or one can use TCEP. It does not reduce Me2+ at all
and is much more stable than DTT. It is expensive but
0.5-1 mM seems to be sufficient.
Dima
I would not recommand the use of guanidine, it will unfold your protein...
Good luck
Alex
Jenny <[EMAIL PROTECTED]> a йcrit :
Hi All,
I'm trying to purify a protein which is expressed in soluble form.It's a
His-tag protein, so when I run His-column, it's really easy to clog.In the
next step size exclusion purification, the protein come off at the void
volume, which suggests that the protein is severely aggregated.Does any one
have any suggestions that I can modify the purification condition so as to
avoid aggregation problem?Or can I add GuHCl during His column purification
so that it won't clog so easily?Thanks a lot.
Jenny
