The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non-denaturing gels. Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-specific competitor or gel concentration and/or composition. We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp I (stimulatory protein I), NF-Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used. Furthermore, we show the influence of glycine concentration on Sp I binding using recombinant glutathione S-transferase-Sp I fusion protein expressed in Escherichia coli.
- Gel retardation