Donga Akhil R, Kumar Sandeep, Rahimi Mehdi, Kumari Manisha, Godhani Chirag M, Soumith Gundlapalli, Solanki Tanvi J, Kumar Kamlesh
Department of Genetics and Plant Breeding, C. P. College of Agriculture, S. D. Agricultural University, Sardarkrushinagar, 385506, India.
Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.
Photochem Photobiol Sci. 2025 Sep 11. doi: 10.1007/s43630-025-00773-0.
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.
电泳迁移率变动分析（EMSA）是一种用于研究蛋白质 - 核酸相互作用的常用且有效的分子生物学工具。EMSA是一种用于揭示蛋白质（如转录因子）与DNA或RNA结合动力学的技术。该技术有多个重要阶段。为制备DNA/RNA探针，需合成一段编码感兴趣的精确核苷酸序列的小DNA或RNA片段，并用放射性或荧光标记物进行标记。然后将标记的DNA/RNA探针与感兴趣的蛋白质在反应缓冲液中进行处理。这通过非变性聚丙烯酰胺凝胶电泳（PAGE）来实现，该电泳可使蛋白质 - DNA/RNA复合物保持完整。电泳后，通常通过将凝胶暴露于X射线胶片以检测放射性探针，或使用检测器扫描以检测化学发光/荧光探针来观察凝胶。凝胶上观察到的条带迁移表明DNA/RNA - 蛋白质复合物的形成，从而证实了DNA/RNA与蛋白质之间的相互作用。分子生物学和生物技术的进步，如蛋白质纯化、核酸标记和检测方法的改进，可间接提高EMSA实验的灵敏度、效率和准确性。如今，放射性同位素的使用正被染料所取代，这能以简单便捷的方式呈现游离DNA片段、结合的DNA片段和结合的蛋白质。
