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).
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
