RNA Electrophoretic Mobility Shift Assay (REMSA):

Increasing amounts of purified protein and labeled probes (10 fmol, see in vitro transcription) were combined in the binding buffer for 30 minutes. The final REMSA binding buffer concentrations were 140 mM KCl, 10 mM HEPES pH 7.9, 5% glycerol, 1 mM DTT and 0.33 mg/ml tRNA. The reaction was further supplemented with 15 μg salmon sperm DNA to reduce non-specific interactions from the lysate. Complexes were resolved on either 4% or 6% non-denaturing polyacrylamide gels. The gels were dried and the appearance of complexes was visualized by exposure to BioMax MR film. Dissociation constants (Kd) were determined by quantified the protein- bound fractions using ImageJ software and plotted against protein concentration (nM). Kd values were extracted from plots fitted to a hyperbolic function in Graph PAD Prism software (OriginLab).