Help with Gel Filtration

James Bassuk bassuk at u.washington.edu
Sun Nov 17 17:56:14 EST 2002


On 26 Oct 2002 dustjohn at hotmail.com wrote:

>
> Trying to separate a 60kDa His-tagged protein from a ~26kDa Ecoli protein that purifies along with it on the His-column.  Had no luck separating the two with a Sephadex G-75 column (bed length ~25cm, column diameter ~1.5cm).  Switched to G-50, same bed length and, again, no separation.  Should I run a longer column (~90cm bed length)?  Should I increase/decrease the filtration rate (usually ~1ml/min).
> Thanks a million!
> Dustin
>
> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0
>

This is easy -- the 26kDa contaminant is a cis-trans prolyl isomerase that
loves metals -- and hence this is why it shows up in your prep.

Here is the workaround:

* the next time you make an extract, add imidazole to a final
concentration of 25 mM to your sample.  this will keep the isomerase from
binding Ni-NTA under regular conditions.

* if that fails then you can do what we do -- an Amersham/Pharmacia
Superdex 70 resin -- 60 cm bed height run at 0.1 ml/min will easily
separate the 2 proteins.

Jim




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