Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA)

Electrophoretic mobility shift assay can be used to identify the protein-DNA interaction (EMSA). The migration of nucleic acid is slower in the complex it forms with the protein than in its free state. The purified protein and DNA ligands are used for EMSA in vitro, with a final volume of 20 μl of binding buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl₂, 10 % glycerol) for 30 min of room temperature incubation. DNA is about 200 bp long and was either directly synthesized or PCR-amplified. Pseudomonas aeruginosa PAO1 genome was used as the template in the PCR reaction. DNA ligand is present in each reaction at an amount of 30 ng, and the protein concentrations are 0 μM, 0.5 μM, 1 μM, and 1.5 μM, respectively. For weak interactions, it is advised to increase the protein concentration. Finally, the reactions were loaded and electrophoresed on a 6 % polyacrylamide gel at 100 V for one hour. The gels were then observed and captured using the gel imaging equipment (Bio-Rad) after being exposed to nucleic acid dye for 5 minutes. In each group of reactions, DNA probes for the negative controls were picked at random and verified to be devoid of the matching TFBSs. At least twice more replications of the experiment produced comparable outcomes.