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

Sarah E. Altschuler, Karen A. Lewis, Deborah S. Wuttke
  • 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
    Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, United States | deborah.wuttke@colorado.edu

Abstract

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.

Keywords

high-affinity, protein, nucleic acid, EMSA, filter binding

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Submitted: 2012-12-19 01:30:15
Published: 2013-09-20 16:46:25
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Copyright (c) 2013 Sarah E. Altschuler, Karen A. Lewis, Deborah S. Wuttke

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