I have attached a picture of a western blot I just ran using human lymphocyte lysates. You'll see that the two strongest bands are very close together. This western was run on a 8-16% gradient gel, but I've also run it on 10% Tris-Glycine gel, and so far I have failed to to adequately separate the bands. What would be the best method for separating these two bands? Should I let electrophoresis run longer at a lower current? Any ideas about what the optimal gel concentration should be?

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Attached file: two bands.jpg (15 downloads, 14KB)

I doubt a 1D gel that can separate proteins that close in molecular weight. The only thing you might try is to run a low % gel until the markers start to run off the gel. However, plenty of people publish Westerns with non-specific bands as long as they are properly controlled for it shouldnt be a problem.

How do you know the upper band is non-specific?

Rb