We did the EMSA as well as fluorescent anisotropy for the same protein-DNA and we observe strong binding in anisotropy study but no band in emsa although very slight smear observed. What could be the possible reason for that??
It could be a question of affinity. To get a band shift in EMSA, the complex has to remain intact while passing through the gel, which causes some dilution and also has a sieving property (like gel filtration). If the affinity is not high enough, the complexes may dissociate once they enter the gel. This is not an issue for the fluorescence anisotropy experiment, which is conducted at equilibrium.
However, fluorescence anisotropy relies on the attachment of a fluorescent dye to the DNA. Since it is possible for the fluorescent dye to interact directly with the protein, rather than the DNA, you should perform a control experiment using unlabeled DNA as a competitor.
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