Semiquantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments. Gamsjaeger, R., Kariawasam, R., Bang, L., Touma, C., Nguyen, C., Matthews, J., Cubeddu, L., & Mackay, J. Analytical Biochemistry, 440(2):178-185, 2013. doi abstract bibtex 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. © 2013 Elsevier Inc. All rights reserved.
@article{
title = {Semiquantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments},
type = {article},
year = {2013},
pages = {178-185},
volume = {440},
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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. © 2013 Elsevier Inc. All rights reserved.},
bibtype = {article},
author = {Gamsjaeger, R. and Kariawasam, R. and Bang, L.H. and Touma, C. and Nguyen, C.D. and Matthews, J.M. and Cubeddu, L. and Mackay, J.P.},
doi = {10.1016/j.ab.2013.04.030},
journal = {Analytical Biochemistry},
number = {2}
}
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