Hi everyone, I'm having a similar issue so I'll use this topic (even if I think the problem may be totally different). When running gels, I'm often having a very narrow horizontal line which often runs at my protein's height. In the first gel (12%), you can see that the horizontal line corresponds to a very bright band of the marker; by counting the the marker's band, it can be noticed that there should five blue bands between the red and green ones, so the bright blue band indicates there are three bands of the marker that are not well-resolved. In the second gel (15%), all the marker's band seems resolved (except fot the High Mw ones) but the protein seems to "focus" on a very narrow line preventing me to see what's in there.

When the problem happens, I noticed that during the run there's a line of different transparency, like if there was some inhomogeneity of the gel there, but this is visible only during the run and not before or after. That's why I think something bad happens during the run. I run gels for 1.5-2h (current starts usually from 70mA and end to 30) and the separation stops when that "transparency line" start to be seen so increasing run time won't be helpful I guess.

I've got this problem for more than the two gel I've posted; I tried to change the voltage (100 to 150V), I used a different batch of bis/acrilamide, I reprepared Tris buffer pH 8.8 and 6.8 (starting from Tris-HCl). My protein samples are in 20mM Tris + 50 to 150mM NaCl and are diluted 1:1 with Laemmli buffer with DTT added fresh.

Any help, idea or opinion would be much appreciated, thanks a lot!

Ale