Help with Gel Filtration

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Sat Oct 26 08:46:34 EST 2002


> Trying to separate a 60kDa His-tagged protein from a ~26kDa Ecoli protein
that purifies along with it on the His-column.

The 26 kDa protein is a cis-trans proline isomerase. It is routinely
enriched in His-tagged preps. Consider reducing your ni-NTA column volume to
minimize nonspecific binding.

>  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.

As a take-home excercise, look up specs for these two resins
Amersham-Pharmacia has a handout on their site). Which one has larger cutoff
? In this light, why was there very little chance that G50 works in your
case ?
Apart from other things, your protein may be actually binding to the
contaminant. Consider using ion-exchange resins or HIC, before going to
sizing.

>Should I run a longer column (~90cm bed length)?  Should I
increase/decrease the filtration rate (usually ~1ml/min).
1 ml/min is a little too fast. 0.5 is better. How much do you load ? You're
not supposed to load samples in above 1.5-3% of the
column active volume.

A.G.E.





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