Work

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

Year: 2013

Type: article

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.

Source: Journal of Nucleic Acids Investigation

DOI: 10.4081/jnai.2013.4646

Authors: Sarah E. Altschuler, Karen A. Lewis, Deborah S. Wuttke

Institution: University of Colorado Boulder

Language: English

Topic: DNA and Nucleic Acid Chemistry

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: diamond

Funders: National Science Foundation, National Institutes of Health, National Institute of General Medical Sciences

Awards: Interdisciplinary Predoctoral Training in Molecular Biophysics, Understanding the Physical Mechanism of Telomere End Capping