Some say that the excess DTT and dust cause the streaking and add a
second euilibration with 2% iodoacetamide in the equilibration step. Or
it could be the incomplete reformation of the S-S bonds after reduction,
which the IAA also takes care of.
> I seem to get blank areas after silver staining my 2D gels - any ideas to a cause? I use a silver diamine stain with 0.83% sodium thiosulphate in the gel matrix.
That is usually caused by having too much protein in that spot.
> What could cause a background 'cloud' near the end of a gel? I have tried reducing the IPG buffer down to 0.5% but still get a smear.
My guess is that it's crap in your sample buffer and/or bromophenol
blue.
> I have read quite a few troubleshooting guides and have not been able to solve the above questions. I am contemplating taking a 2D course. However, the only two I have found are in Europe. Does anyone know of a 2D electrophoresis course in North America?
Sorry can't help you there.
-- Regards, Mustafa Unlu University of Aberdeen Proteomics Facility