IEF gels

Duncan Clark Duncan at genesys.demon.co.uk
Tue May 30 10:50:27 EST 1995


Hi Folks,

I've been running IEF gels of various recombinant thermophilic DNA 
pols but keep finding that some refuse to run as single bands. The 
enzymes are homogeneous on SDS gels with silver staining. On IEF 
gels, either native or denaturing with 8M Urea I get one main band 
and several others (10% intensity each) over roughly 0.5 pI units 
either side (staining with coomassie blue). I am discounting 
proteolysis in that the known effect of E.coli proteases on these 
pols would show up on SDS gels. Any proteolysis would also have a 
marked effect on activity which I'm not seeing. 

Could it be some sort of aggregation? I've thought of treating 
the samples with an SDS cracking buffer as per SDS PAGE before 
loading onto a denaturing IEF but have yet to try it. What happens if 
one puts SDS in an IEF gel, concn of SDS?

All suggestions welcome.

Duncan



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