Hello everyone,
I have performed EMSA experiment with an RNA-binding protein and RNA. For both ssRNA and dsRNA, for higher concentration of protein, I observe a high intensity band at the dye front (lower than the free RNA band). The intensity of this band decreases with decrease in protein concentration. I have performed the experiments in a cold room with 0.5X TAE buffer and 8% gel.
Any idea why this is happening?
In an Electrophoretic Mobility Shift Assay (EMSA), the mobility of RNA-protein complexes (RNP-RNA) and free RNA is affected by various factors including size, charge, and shape. Here's why RNP-RNA might migrate faster than free RNA at higher protein concentrations . the combination of changes in size, charge, shape, and gel interaction due to the formation of RNP-RNA complexes at higher protein concentrations can lead to faster migration of the complexes compared to free RNA during EMSA.
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