ekhatipo at NOSPAMmidway.uchicago.edu
Wed Nov 29 12:50:35 EST 2000
I believe you are not overloading your gels with protein, as well as you are
not using old precast or hand-made gels that may have gaps between the gel
and the glass walls, as well as you are using a good staining solution that
still have a fixing agent (acid), do you?
"Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
news:Pine.SGI.3.96.1001128131837.12641681A-100000 at mole.bio.cam.ac.uk...
> > Since PEG is not charged and would not co-polymerize with AA,
> > I can't see how trace amounts of PEG (the only kind possible as
> > far as water supply contamination is concerned) can
> > affect PAGE behavior appreciably.
> > I think you need to look elsewhere. What does "gels go bad"
> > mean exactly? What do you observe?
> > - Dima
> Dear Dima, the gels have protein bands which are really smeary upon
> staining. Having read the article recommended by Harold (thanks again
> Harold), there are some clues that might interest you:
> An effect of polyethylene glycol 8000 on protein mobility in sodium
> dodecyl sulfate polyacrylamide gel electrophoresis and a method for
> eliminating this effect. Odom,O.W., Kudlicki,W., Kramer,G., Hardesty,B.
> Analytical Biochemistry 245:249-252
> p249 "The mobilities of the three largest proteins (in the standard
> proteins used) increase with PEG, while those of the two smallest proteins
> p249 "It is seen that those proteins behind the PEG move front move
> faster than normal, while those ahead of the PEG move more slowly".
> Which is similar to what I see, but I get much more smearing.
> p250 "Kurfurst reported that the PEG migrates during PAGE only in the
> presense of SDS this is undoubtedly due to the fact that PEG binds to
> the SDS micelle".
> (though PEG can also cause anomolies in DNA gels which contain no SDS!)
> p250 the authors reduced the formation of PEG/SDS micelles and
> the bad PEG effect by reducing the concentration of SDS from 0.1 to 0.05%
> and tris from 125 t 93.8mM.
> p250.-1.4 "It is still unclear exactly how the PEG-micelle complex affects
> protein mobility...".
> Any ideas out there? Mike.
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