Help with Gel Filtration

Chris Chang cchang at chem.ufl.edu
Mon Oct 28 16:24:35 EST 2002


Dextran-based resins like Sephadex also interact with proteins via hydrogen
bonds, which can interfere with separation, particularly in low ionic
strength buffers. I'd also suggest trying the technically more
straightforward ion exchange or HIC chromatographies, but if you must stick
with gel filtration, make sure you have a little ionic strength--0.1 M KCl
or NaCl is fine. On that note, changing conditions on the nickel column
might obviate the need for the additional column altogether...

CHC

<dustjohn at hotmail.com> wrote in message
news:20021026004236.28950.qmail at ww02.hostica.com...
>
> 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
>
>
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