In article <Pine.GSO.3.95.970325205643.4983A-100000 at planetx.bloomu.edu>,
"Thomas N. Dentel" <tndent at planetx.bloomu.edu> wrote:
> On Wed, 19 Mar 1997, Aine L. Plant wrote:
>> > We have been running 2-D gels for approx. 2 years using the protean 2-D
> > gel system. Up until now the gels have been running well, however,
> > recently we have started to periodically obtain gels that have
> > substantial amounts of smearing. This happens about every 20 gel runs
> > and appears to be a problem with the SDS-PAGE rather than the ief since
> > 1) it only applies to a few tubes out of the total run for ief and 2)
> > the markers are also smeared. We have checked our solutions and are
> > careful when casting gels.
> >
> I am hardly an expert, just getting into electrophoresis myself,
> but I have recent (like today) experience with smeared gels. My experience
> is that if you have cellular crud in the homogenate that gets onto the
> wickes (assuming your set up uses wicks) you get lots of smearing.
>Hi,
I got this problem too sometimes. I use immobilines gels but your problem
should be of the same nature. It's a problem of proteins solubility.
Increase the concentration of urea in the equilibration buffer (up to 6M).
The equilibration buffer should also contain glycerol (10 to 20%), DTT (10
mM) and 2% SDS. The first dimension gel can be equilibrated 2x 10 min in
equilibration buffer.
But that's not all. You may face also a problem of unsufficient
concentation of SDS in the upper tank during the 2nd dimension
electrophoresis. Maybe you run too many gel at a time with the same
electophoresis apparatus and the SDS gets eluted after a few hours. To
overcome this problem, you may double the concentation of SDS in the upper
buffer tank (0.2% instead of 0.1%) and/or renew the upper running buffer
when migration is half completed.
Finally, it is also a good idea, once the bromophenol blue has entered the
2nd dimension gel, to discard the 1st dimension gel. This will eliminate
the streaking of some major proteins. Without to mention also the
importance of the current and temperature setting during the 2nd dimension.
Hope this helps a little
--
Didier Thomas
Dept. of Physiology and Biophysics
Med. Sci. I, Room D340
University of California, Irvine
Irvine, CA 92697
