Electrophoretic mobility shift assay for characterizing RNA-protein interaction.
Electrophoretic mobility shift assay, or EMSA, is a well-established technique for separating macromolecules under native conditions based on a combination of shape, size, and charge. The use of EMSA can provide both general and specific information concerning the interaction between two macromolecules such as RNA and protein. Here we present a protocol for the practical use of EMSA to assess protein-RNA interactions and ribonucleoprotein (RNP) assembly. The conceptual framework of the assay is discussed along with a step-by-step procedure for the binding of archaeal ribosomal protein L7Ae to a box C/D sRNA. Potential pitfalls and common mistakes to avoid are emphasized with technical tips and a notes section. This protocol provides a starting point for the design and implementation of EMSA in studying a wide variety of RNP complexes.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA. ktgagnon@gmail.com
Keith T
KT
Gagnon
E Stuart
ES
Maxwell
D019843:Archaeal Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D012261:Ribonucleoproteins; D012313:RNA