Help with Gel Filtration

kaj.stenberg at kaj.stenberg at
Sat Oct 26 16:28:13 EST 2002

dustjohn at 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).

It is a dimer in your buffer system. If you insist, try Superdex-200, or
depending on amounts needed, HPLC. Adding (fresh) 1-10mM DTT to the 
buffer could make wonders. 
	Separating 56kd from 60kd is not a gel filtration aproach.

Kaj Stenberg

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