2D electrophoresis headache!

[Kajiwara Hideyuki]

Thomas wrote:
> 
> In article <63lh4t$a8t$1 at svr-c-02.core.theplanet.net>, "Andrew Pridmore"
> <andrew at apridmore.source.co.uk> wrote:
> 
> > I have been attempting obtain whole-cell protein profiles of the oral
> > anaerobe Porphyromonas gingivalis using two-dimensional electrophoresis,
> > basically according to the method of O'Farrell (1975). I get clear bands in
> > the first dimension (isoelectric focussing in tube gels) but these proteins
> > fail to transfer into the second dimension slab gel. I have varied the
> > concentration of SDS, pH and incubation time in the second dimension buffer,
> > and ensured good contact between the two gels. None of these variables
> > produce an improvement.
> >
> > 2D electrophoresis is not known for being straightforward, but does anyone
> > have any experience of these problems and any suggestions for improving my
> > results?
> 

In my experience, 1st dimension should be done in tube gel.  The 2nd
dimension was norml SDS-PAGE.  The low-conc. acrylamide gel can be put
out from tube.  The tube gel was placed on the top of the stacking gel
of SDS-PAGE after shaking at room tenmp for 10 min in Sample O
solution.  The tube gel can be immoblixed by agarose.
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   Hideyuki Kajiwara
   National Institute of Agrobiological Resources
   Dept. of Biotechnology, Lab. of Gene Engineering
   E-mail: kajiwara at abr.affrc.go.jp
   Fax: 81-298-38-7073
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