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05-08-2006 10:42 AM

Native PAGE question
 

Hi all,

I have a doubt about native PAGE gel preparation and will greatly apreciate any help from
you "protein expert" guys :-)

I am doing 7% native acrylamide gels to separate two isoforms of glutamine synthetase (GS)
from plant crude extracts and then stain the activity in-gel before coomassie. My problem is that
one isoform is unstable and inactivates during the electrophoresis (I normally run it overnight
in a cold room) so I am looking for some way to improve its stability during the run.

As the enzyme in the crude extracts is stabilised by DTT, glycerol and glutamate (that is a GS substrate)
I was thinking if is it possible to add this compounds in the gel! Does anyone knows if this compounds
can be added to the native PAGE gel and /or loading buffer???

Thanks a lot for your help,

Marco Betti Ph.D.
Departamento de Bioquímica vegetal y Biología Molecular
Universidad de Sevilla, Spain


nightfirecat@gmail.com 05-09-2006 07:21 AM

Native PAGE question
 
Are there any disulfide bonds in your GS protein?

DTT always reduces the oxidized disulfide bonds, which could destroy
the confirmation of the protein, even its biological activity.



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