TY - JOUR
T1 - Semi-quantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments
AU - Gamsjaeger, Roland
AU - Kariawasam, Ruvini
AU - Bang, Line H.
AU - Touma, Christine
AU - Nguyen, Cuong D.
AU - Matthews, Jacqueline M.
AU - Cubeddu, Liza
AU - Mackay, Joel P.
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
UR - http://handle.uws.edu.au:8081/1959.7/529468
U2 - 10.1016/j.ab.2013.04.030
DO - 10.1016/j.ab.2013.04.030
M3 - Article
SN - 0003-2697
VL - 440
SP - 178
EP - 185
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -