Using Fluorophore-labeled Oligonucleotides to Measure Affinities of Protein-DNA Interactions

Overview

Journal Methods Enzymol

Specialty Biochemistry

Date 2009 Jan 21

PMID 19152864

Citations 55

Authors

Brian J Anderson

Chris Larkin

Kip Guja

Joel F Schildbach

Affiliations

Soon will be listed here.

Abstract

Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with a variety of labeled oligonucleotides and the relaxase domain of F plasmid TraI. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

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