Problems w/ native PAGE

Yasha Hartberg Yasha at bioch.tamu.edu
Mon Oct 24 19:28:05 EST 1994


In article <bucc0003.783012079 at gold.tc.umn.edu>, bucc0003 at gold.tc.umn.edu
(Paul A Bucciaglia) wrote:
> I have been having problems with native PAGE of proteins.  When I extract 
> total proteins from tobacco flower organs or leaves (I've listed my 
> protocol below) and run them on 4 to 20% denaturing SDS gels, I get fine 
> resolution of bands with no detectable smearing.  If I run the same 
> amount (30 ug) on a 15% native gel, the bands are poorly resolved and 
> smeared along the edges of each lane.  I have also noticed a band of 
> precipitated proteins on the interface of the stacking and resolving 
> gels; this occurs weather I layer the resolving gel with butanol OR water 
> so I don't believe residual butanol is the main problem, as was mentioned 
> earlier on the net. I also briefly centifuge the preps after adding 
> loading buffer, so I don't believe that particulates are to blame.

The problem is with the precipitated protein you see at the interface.  It
is not uncommon to see this type of smearing.  The precipitated protein
slowly redissolves as electrophoresis goes on so that protein is released
slowly into the resolving gel.  My guess is that SDS prevents aggregation
and precipitation so that you don't see the smearing on the denaturing
gels.

I have noticed that this problem is more pronounced in samples that contain
both  BMe and EDTA, even more pronounced in samples with DTT and EDTA.  In
addition, your concentration of BMe seems a bit high to me.  I would
recommend playing around with your extraction buffer and see if the
problems go away.

Yasha Hartberg
Texas A&M University
"If people want to spend their whole lives creaming and tweezing and
brushing and tilting and gluing, that's really okay too, because it gives
them something to do."  Andy Warhol



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