Hi, I am assuming that you are using double-stranded DNA probes.

If that is the case, three possibilities come to mind:

If you are using a nuclear or cell extract, the transcription factor might still be bound to its target sequence in the extract, so you might need a DNA-free extract or purified protein fraction. Secondly, the salt conditions of the protein sample you are using will strongly influence DNA-binding or non-binding. Finally, many transcription factors will only bind as part of a protein complex that requires longer DNA fragments (100 bp or even significantly more).

@Sven Buelow, Thanks for your reply. How can I make the DNA dissociate from from the nuclear extract? and how can I decide the salt concentration of protein sample. I am not very sure about the required salt concentration of my protein-DNA binding yet.

Hi,

EMSA is not an equilibrium method, as during electrophoresis DNA is spearated from protein and binding equilibrium shifts to dissiciation. Also, once in a gel, the environment of your complex changes drastically. It is not a problem for strong complexes, but if the complex is weak or has fast dissociation rate, it might not be detectable in gel. The suggestion about DNA already bound is also very plausible. You can try binding reactions in a buffer without monovalent salts. Also, make sure to include BSA in your binding reaction to prevent loss of protein due adsorption on the tube.

Thanks for Ruta, I increased the labeled DNA again and added EDTA in the binding buffer, some weak bands did occur. But there are several bands. You can see that in the picture. Now I am confusing how can I know which band is I want? should I add more poly IC next time?

Now it seems that you added a lot of salt, as your DNA only bands are compressed. Provided that you can rule out the presence of nuclease, it seems that all your DNA is bound. the lowest band that you've marked looks like salt front (or poly IC?) front and most likely is an artefact. you can check this by making you "DNA only" sample the same composition as others. The complex you want is either smeared throughout the lane or in the upper bands. The latter is more likely as they seem to be sensitive to unlabelled competitor. It is also worth titrating the protein starting from lower concentrations. If my scenario is correct, you have a huge excess of protein binders.

Thanks Ruta, I prefer the higher band is specific. I run again and got the same result. Now I am confused if I want to know what protein is in this band that binds to DNA with allele A rather than G. What should I do? It's a AT rich sequence, many transcriptional factors can bind to it. But I just want to know the protein in that specific band. Do you have some suggestions? Thanks.