gelfiltration on superdex 75 pg

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Fri Sep 29 03:51:41 EST 2000


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.

If you want to use salt, the maximal concentration mostly depends
on your protein: is it still stable under high salt conditions?
(It does not necessarily have to be active as long as activity
recovers when you remove the salt.)

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */






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