Unraveling biomolecular interactions: a comprehensive review of the electromobility shift assay

Abstract

The electromobility shift assay (EMSA) is a popular and productive molecular biology tool for studying protein-nucleic acid interactions. EMSA is a technique applied to the revelation of the binding dynamics of proteins, like transcription factors, to DNA or RNA. There are ample essential phases in the technique. To make the DNA/RNA probe, a small DNA or RNA fragment encoding the exact nucleotide sequence of interest is synthesized and labeled with a radioactive or fluorescent marker. The labeled DNA/RNA probe is then treated in a reaction buffer with the protein of interest. This occurs through non-denaturing polyacrylamide gel electrophoresis (PAGE), which allows the protein-DNA/RNA complexes to remain intact. After electrophoresis, the gel is visualized, typically by exposure to X-ray film for radioactive probes or scanning with a detector for chemiluminescence/fluorescent probes. A shifted band observed on the gel signifies the establishment of DNA/RNA-protein complexes, thereby confirming the interaction between the DNA/RNA and the protein. Advancements in molecular biology and biotechnology, such as improvements in protein purification, nucleic acid labeling, and detection methods, can indirectly enhance the sensitivity, efficiency, and accuracy of EMSA experiments. Nowadays, use of radioisotopes is being replaced by dyes which results in free DNA fragments, bounded DNA fragments, and bounded proteins in a simply convenient way.

Keywords: Electrophoretic mobility shift assay (EMSA); Nucleic acid–protein binding; Protein–DNA interactions; Transcription factors.