https://doi.org/10.4081/jnai.4646

Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

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Submitted: 19 December 2012
Accepted: 7 February 2013
Published: 20 September 2013
high-affinity, protein, nucleic acid, EMSA, filter binding
Abstract Views: 4370
PDF: 1362
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Authors

Sarah E. Altschuler
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, United States.
Karen A. Lewis
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, United States.
Deborah S. Wuttke
deborah.wuttke@colorado.edu
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, United States.
The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

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Supporting Agencies

National Institutes of Health (National Institute of General Medical Studies) and the National Science Foundation

How to Cite

Altschuler, S. E., Lewis, K. A., & Wuttke, D. S. (2013). Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions. Journal of Nucleic Acids Investigation. https://doi.org/10.4081/jnai.4646
Copyright (c) 2013 Sarah E. Altschuler, Karen A. Lewis, Deborah S. Wuttke Creative Commons License

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