Help on Gel Filtration

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Mon Mar 23 02:49:21 EST 1998


In article <01IUZHD6A7WK9ODSFN at cc.usu.edu>, slzmz at CC.USU.EDU (Xie Yi) wrote:

> Hi!
> I am trying to purify a small protease, which has the size about 13KDal. 
> What I did is that first I ran the gel filtration on Superose 12, and 
> then I collected the peak corresponding the activity peak. But when I run 
> the SDS-PAGE, I found I got a mixture of proteins. Even some pretty big 
> protein like 70KDal is appearing in the elution position of 13KDal. What 
> I did wrong?

Gel filtration is not usually used as the first chromatographic step in
the isolation of a protein.

Having said that, if the size exclusion limit of Superose 12 is 12 kDa
then it is no wonder that your activity peak is contaminated by large
proteins; they likely were coming out close to the void volume where the
first fractions of your 13kDa protein would come out. If you still wish to
use gel filtration as a first step then try choosing a higher exclusion
limit say 25 or 50 kDa; proteins larger than this should come out in the
void volume and your protein, being smaller, should be retarded by the
matrix and come off much later.

Just some thoughts.

Cheers

Karl the hepB guy



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