Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA)

The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction base d on the observation that binding of protein to DNA or RNA fragments usuall y leads to a reduction in the electrophoretic mobility of the fragment in n on-denaturing gels. Here we report on the critical role of the running buff er and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-sp ecific competitor or gel concentration and/or composition, We demonstrate d ifferences in the binding patterns obtained with oligonucleotides containin g binding sites for the ubiquitously expressed transcription factors Sp I ( stimulatory protein I), NF-Y (nuclear factor Y) and USF (upstream stimulato ry protein), which are dependent on the ionic strength of the running buffe r used. Furthermore, we show the influence of:glycine concentration on Sp I binding using recombinant glutathione S-transferase-Spl fusion protein exp ressed in Escherichia coil.