Help with Gel Filtration
res0khk2 at verizon.net
Sat Nov 2 02:58:32 EST 2002
>From the information provided I would not immediately assume dimerization
via a disulfide bond. If you do come to this conclusion make sure you
dialyze or add neat EDTA to ~10-20 mM before trying to reduce your sample.
<kaj.stenberg at helsinki.fi.invalid> wrote in message
news:apf1dd$lhs$1 at oravannahka.helsinki.fi...
> 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
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