2D electrophoresis headache!
kajiwara at abr.affrc.go.jp
Fri Nov 14 19:56:13 EST 1997
> 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.
National Institute of Agrobiological Resources
Dept. of Biotechnology, Lab. of Gene Engineering
E-mail: kajiwara at abr.affrc.go.jp
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