I have recently run IEF (7cm, PH 3-11, NL) and 2D gel electrophoresis (12.5%). After CBB staining, the dots were not shown as single dots. Most of them are tailed (see attached). I would guess there is something wrong with the IEF step. Any idea about this?
Tailed the wrong way for an IEF issue: isoelectric focussing is the first dimension (left/right), so problems with IEF result in horizontal streaking rather than vertical streaking.
The vertical streaking is interesting: suggests each protein exists as multiple discrete species with slightly different molecular weights. Easiest explanation for this would be some sort of post-translational modification (my suspicion would be user-generated, due to the ubiquity: have you treated your samples with any covalent-modification agent?).
Thanks John! I agree, it is likely due to the 2nd gel run, not IEF. However, we didn't do any treatment with the samples. We have before run this several times using the same settings, everything was right. This problem just recently showed up. Will do trouble shooting with the 2nd SDS PAGE to see what is wrong.
It might come from the gel preparation and the running buffer. Good luck
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