eluting proteins from gels

Dennison dennison at unpsun1.cc.unp.ac.za
Mon Jun 1 12:09:14 EST 1998

> > Recently, i have been dabbling in a bit of protein purification and on
> > a
> > coomasie gel i have only a few proteins, one in particular is stronger
> > than
> > the others. I wish to show that this stronger band is the protein
> > responsible for activity I am detecting in the fraction. Is it
> > feasible to
> > simply elute this band from the SDS gel and assay this for activity,
> > i.e.
> > is there a simple way of eluting the protein directly from the gel
> > with a
> > good chance of renaturing the protein?
> >
> > Thanks in anticipation
> >
> > Karen Spink
> >
> > E. mail pdxkgs at pdn1.gene.nottingham.ac.uk

We work with proteinases (cathepsins) and we have had no luck in trying
to elute activity from polyacrylamide gels.
However, gelatin zymograms work well.  Gelatin is included in the gel
and SDS run in the usual way, except protein is not boiled.  After
running, the SDS is removed with a triton-X wash and activity is
revealed as a clear area upon staining of the gelatin.  See Heussen and
Dowdle, Anal. Biochem. (1980) 102, 192-202.  
The point is that the activity is there but for some reason cannot be
successfully eluted from the gel.  The proteins themselves are
successfully eluted, as evidenced by immunoblotting, but they must be
screwed up during elution.

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