The gel electrophoresis mobility shift assay (EMSA) is a widely used method for detecting protein-nucleic acid interactions. It involves combining solutions of protein and nucleic acid, followed by electrophoresis under native conditions through polyacrylamide or agarose gels. The distribution of species containing nucleic acid is then determined, typically by autoradiography of 32P-labeled nucleic acid. Protein-nucleic acid complexes generally migrate more slowly than free nucleic acid. This article discusses the key factors affecting the stability and electrophoretic mobility of complexes, provides a representative protocol, and addresses common issues and troubleshooting strategies. EMSA is sensitive and versatile, suitable for a wide range of nucleic acid sizes and structures, and can be used to detect binding stoichiometries, affinities, and kinetics. However, it has limitations, such as the non-equilibrium nature of the assay and the inability to directly determine protein molecular weights or identities. Alternatives to EMSA, such as filter binding and footprinting assays, are also discussed, highlighting their advantages and disadvantages. The article emphasizes the importance of optimizing nucleic acid target design, binding conditions, and electrophoresis conditions to achieve optimal results.The gel electrophoresis mobility shift assay (EMSA) is a widely used method for detecting protein-nucleic acid interactions. It involves combining solutions of protein and nucleic acid, followed by electrophoresis under native conditions through polyacrylamide or agarose gels. The distribution of species containing nucleic acid is then determined, typically by autoradiography of 32P-labeled nucleic acid. Protein-nucleic acid complexes generally migrate more slowly than free nucleic acid. This article discusses the key factors affecting the stability and electrophoretic mobility of complexes, provides a representative protocol, and addresses common issues and troubleshooting strategies. EMSA is sensitive and versatile, suitable for a wide range of nucleic acid sizes and structures, and can be used to detect binding stoichiometries, affinities, and kinetics. However, it has limitations, such as the non-equilibrium nature of the assay and the inability to directly determine protein molecular weights or identities. Alternatives to EMSA, such as filter binding and footprinting assays, are also discussed, highlighting their advantages and disadvantages. The article emphasizes the importance of optimizing nucleic acid target design, binding conditions, and electrophoresis conditions to achieve optimal results.