Help on Gel Filtration

Cornelius Krasel krasel at
Tue Mar 24 08:42:14 EST 1998

Xie Yi <slzmz at CC.USU.EDU> wrote:
> 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?

This is difficult to tell unless you give more information. For example,
what is the material to start from, how large is the proportion of your
protease in the complete sample, etc.

As others in this thread have already mentioned, gel filtration is not
a good method to start a purification. Big proteins can appear in late
fractions on a gel filtration if they interact somehow with the gel
matrix. You can sometimes prevent this by including more salt in the
running buffer.


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

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