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?

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

I wish you might have shorted out the problem , if not
then ..
kindly clear me few things so that I can troubleshoot this problem.
They seem to me ERK 42/44?
Are you running minigels?
Run you dye front till the edge. or lil over run if you don't have anything to do with your lower molecular weight protein.
because even with 15% ERK42/44 get separated easily.