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

Roland Gamsjaeger, Ruvini Kariawasam, Line H. Bang, Christine Touma, Cuong D. Nguyen, Jacqueline M. Matthews, Liza Cubeddu, Joel P. Mackay

    Research output: Contribution to journalArticlepeer-review

    17 Citations (Scopus)

    Abstract

    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.
    Original languageEnglish
    Pages (from-to)178-185
    Number of pages8
    JournalAnalytical Biochemistry
    Volume440
    Issue number2
    DOIs
    Publication statusPublished - 2013

    Fingerprint

    Dive into the research topics of 'Semi-quantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments'. Together they form a unique fingerprint.

    Cite this