I am also doing EMSA with RNA probe. I encountered the same problem which my shifted band seems stuck in the well. Since you mentioned that is also considered as binding, I am kind of wondering how can I make the stuck band move in the gel, are there any other way rather than lowing the percentage fo the gel? I will really appreciate your thought on this.
Originally Posted by kevin_a
Typical DNA EMSA use a 5 or 6% native gel with ~20bp double stranded probe.
Your's is half the mass, so doubling will help, but you'll probably still see diffusion admin pointed out.
You can't just increase the percentage of gel because your protein
robe complexes are going be very high up on the gel - possibly even look stuck in the well. If you are looking for binding vs no binding it may not matter as much, but if you need to resolve multiple bands you will probably want to tweek with the gradients.