title: Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions.
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,Schizo -saccharomyces pombePot1 andSaccharomyces cerevisiaeCdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Altschuler
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Lewis
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA.
authors.forename: Deborah S
authors.initials: DS
authors.lastname: Wuttke
keywords: EMSA; filter binding; high-affinity; nucleic acid; protein