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Biotechnology and Applied Biochemistry (2001) 33, (209–214) (Printed in Great Britain)

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

Karim Roder* and Michael Schweizer†¹

*Department of Nutritional Sciences, University of California, Berkeley, CA 94720, U.S.A., and †Department of Biological Sciences, Heriot-Watt University, Edinburgh EH14 4AS, U.K.

Abbreviations used: AP1, activating protein 1; EMSA, electrophoretic mobility-shift assay; GST, glutathione S-transferase; NF-Y, nuclear factor Y; Sp1, stimulatory protein 1; USF, upstream stimulatory protein.

¹ To whom correspondence should be addressed (e-mail M.Schweizer@hw.ac.uk).

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 Sp1 (stimulatory protein 1), 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 Sp1 binding using recombinant glutathione S-transferase–Sp1 fusion protein expressed in Escherichia coli.

Received 15 January 2001/19 March 2001; accepted 23 March 2001

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