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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