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
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
Thanks for your advice Cornelius !
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