Label-free horizontal EMSA for analysis of protein-RNA interactions

Abstract

We describe a method to analyze the affinity and specificity of interactions between proteins and RNA using horizontal PAGE under non-denaturing conditions. The method permits tracking of migration of anionic and cationic biomolecules and complexes toward anode and cathode, respectively, therefore enabling quantification of bound and free biomolecules of different charges and affinity of their intermolecular interactions. The gel is stained with a fluorescent intercalating dye (SYBR®Gold or ethidium bromide) for visualization of nucleic acids followed by Coomassie® Brilliant Blue R-250 for visualizations of proteins; the dissociation constant is determined separately from the intensity of unshifted and shifted bands visualized by each dye. The method permits calculation of bound and unbound anionic nucleic acid and cationic protein components in the same gel, regardless of charge, under identical conditions, and avoids the need for radioisotope or fluorescent labeling of either component.

Keywords: Dissociation constants; EMSA; RNA; RNA-binding proteins.