The second (upper) shift band in the sample lanes can be due to your protein of interest forming dimers or multimers under the salt conditions of your EMSA, or the upper band may be the band resulting from your protein of interest bound not only to the DNA but also to another protein more or less specifically. Many transcription factors act in conjunction with a second protein (this possibility applies if you are not working with a highly purified transcription factor).

The nature of the upper DNA band in the control is difficult to discuss without knowing how you prepared your DNA probe sample.

Hi Steven, thank you for your answer. But my concern is about lanes 1 and 3 where I am getting an unexpected band. Once we are able to explain that, we can determine the nature of bands in the samples.

Thanks!

Since you haven't provided more detail on the probe and its preparation, but you say it is gel-eluted and sequence-checked: if you haven't done anything with it in-between, the band may indicate a) secondary folding structure (should be checkable based on the known sequence of the probe, whether hairpin structures can occur), or b) dimerization, which again requires sequence features for secondary structures or longer stretches of individual bases or similar. Whenever you have such a sequence it will alway create an equilibrium between the possible forms in solution. So, even after elution from a single band, you should always get two bands again and again, if the sequence is actually the underlying cause. But this also means that the second band should have been existent on the gel you used for elution in the first place, if the salt and pH conditions were not too different. So, you might want to revisit the picture of the original gel or cut out the single band and run it again...