gelfiltration on superdex 75 pg

[Sigrid Van Boxstael]

"Richard P. Grant" wrote:

> In article <39D36691.D21C4C49 at vub.ac.be>, Sigrid Van Boxstael
> <svboxsta at vub.ac.be> wrote:
>
> > I can only recuperate 1.5 mg in the obtained peak.
> > 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.
> > I presume that my protein sticks on the column.
>
> Is the column calibrated?  Is the 'obtained peak' in the correct place -
> are you *sure* it's not running in the void volume?  Where do you see
> the contaminating protein?

I calibrated the column and the protein is coming out on the right position
and
when I put it on native gel it also looks fine.
There are two peaks a very small one with contaminating proteins and a
'bigger' one
with my protein.

>
> If a fraction of your protein aggregates it could barrel through and
> come out first - with you thinking it's the main peak.
>
> --
> Richard P. Grant MAD Phil      http://www2.mrc-lmb.cam.ac.uk/personal/rpg/
>
> Please reply to rpg 'at' mrc-lmb.cam.ac.uk'