gelfiltration on superdex 75 pg

Sigrid Van Boxstael svboxsta at vub.ac.be
Fri Sep 29 06:41:24 EST 2000


Cornelius Krasel wrote:

> Sigrid Van Boxstael <svboxsta at vub.ac.be> wrote:
> > I'm trying to purify my protein.
> > I bring 10 mg (already 90 % pure) on a Pharmacia superdex 75 pg column
> > and I can only recuperate 1.5 mg in the obtained peak.
>
> This happens (unfortunately). Your conclusion seems correct.
>
> > The buffer I used : Tris 20 mM pH 8.2, 2 mM mercapto-ethanol, 150 mM
> > NaCl.
> > When I clean the column after the run there is coming protein of it.
>
> How do you clean the column? Do you use high salt, or are you using
> denaturing conditions? In the latter case, adding salt to the buffer
> will probably not help the recovery rate. You might consider using
> low concentrations of a detergent with a high CMC instead.
>

I use the conditions recommended by Pharmacia to clean the column :
0,5 M NaOH and 0.1 M HCl so quite denaturing conditions.
It probably would have been a better idea if I had washed the column with high
salt.

I mailed  Pharmacia's product specialist and he advised me to
take a  little bit of resin, put it in an eppendorf,  mix it with  my buffer and
a little bit of my protein.
And this for several different salt concentrations.  Afterwards measuring the
activity in the supernatans and see for what salt concentration  I don't loose
activity.

Thanks for your advice Cornelius !

> Sigrid








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