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Anal Biochem. 2013 Sep 15;440(2):178-85. doi: 10.1016/j.ab.2013.04.030. Epub 2013 May 31.

Semiquantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments.

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School of Science and Health, University of Western Sydney, Penrith, NSW 2751, Australia.


One method commonly used to characterize protein-DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein; the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested; this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.


Competition experiments; Kinetics; Protein–DNA interaction; SPR

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