It sounds very much like your running buffer for the 2nd dimension was
inadvertently made up at a lower ionic strength than usual.
This happened to us years ago with a one-dimensional gel and had the same
effects you describe: lowered current flow (because the resistance in the
running buffer is much higher at lower ionic strength), and much slower
runs, i.e., in the "normal" amount of time, much less migration (because the
speed of migration through the gel is proportional to the voltage drop
_across the gel_, and with the higher resistance in the part of the current
flow path through the running buffer, most of the voltage potential
difference you set at the power supply is actually occurring outside the gel
in the running buffer, meaning that the actual voltage drop in the gel
itself is much smaller than usual for the given "Voltage" setting at the
power supply). Can't speak to the wavy brown front, since in our instance we
were running a one-D gel and didn't see this; however, the distorted SDS
front which runs ahead of most proteins in an SDS-PAGE gel probably didn't
run off the gel (due to the same overall slower migration speed) and might
be contributing to what you observed.
---------------------------------------------------------------------------
Bruce A. Stanley, Ph.D.
Director, Scientific Programs
Section of Technology Development & Research Resources, Room C1734
Penn State College of Medicine H093
500 University Drive
Hershey, PA 17033-2390
Voice (717) 531-5329 --- Lab and FAX (717) 531-4055
bstanley@psu.edu
http://www.hmc.psu.edu/stanley/
on 8/31/00 16:52, Di Barraclough at dbarraclough@hort.cri.nz wrote:
> Hello
>
> 2D gel problems:
> My two SDS gels ran at a quarter of the current I'd usually expect. It
> took three times as long as usual for the dye front to come to the end
> of the gel.
> On silver staining one gel, it showed very nicely focussed spots
> clustered together in the top third of the gel, and a distorted wavy
> brown front at the bottom (about where the dye front eventually got to).
> This front is only under where the IEF strip was placed - the MW markers
> are nice and clear.
> This makes me wonder if it is an equilibration buffer contaminant
> problem. The MW markers are perfect.
> My question then is this: would oxidised DTT in the equilibration buffer
> cause such an effect?
> Any advice would be greatly appreciated.
> Regards
> Di Barraclough
> Postharvest Group
> HortResearch
> Auckland
> New Zealand
>
>
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