Chapter 17
Analysis of Protein–DNA Interactions Using Surface
Plasmon Resonance and a ReDCaT Chip
Clare E. M. Stevenson and David M. Lawson
Abstract
The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such
as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR)
is ideally suited for the measurement of these interactions because it is quantitative, simple to implement,
reproducible, can be automated, and requires very little sample. This typically involves the direct capture of
biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the
interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without
damaging the chip surface. Moreover, if the protein–DNA interaction is strong, then it may not be possible
to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly,
this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA
Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times
making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a
simple binding experiment, and analysis of data will be described in detail. Some additional applications will
also be introduced.
Key words SPR, Protein, DNA, Reusable, Binding, Affinity, Kinetics
1 Introduction
SPR is an ideal technique for the study of protein–DNA interactions. It offers several advantages over more traditional in vitro
methods such as electrophoretic mobility shift assays or nitrocellulose filter binding assays. It can provide real-time monitoring of
binding and dissociation events, enabling the identification of binding and the measurement of affinity and kinetic parameters [1–3].
Furthermore, the technique is quick, sensitive, uses low amounts of
protein, and is highly automatable.
A traditional approach to study protein–DNA interactions by
SPR is to tether the DNA (the “ligand”) via a biotin tag to a
streptavidin (SA)-coated chip and then to flow the protein (the
“analyte”) over the top to monitor the interaction. However,
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
369
Analysis of Protein–DNA Interactions Using Surface
Plasmon Resonance and a ReDCaT Chip
Clare E. M. Stevenson and David M. Lawson
Abstract
The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such
as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR)
is ideally suited for the measurement of these interactions because it is quantitative, simple to implement,
reproducible, can be automated, and requires very little sample. This typically involves the direct capture of
biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the
interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without
damaging the chip surface. Moreover, if the protein–DNA interaction is strong, then it may not be possible
to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly,
this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA
Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times
making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a
simple binding experiment, and analysis of data will be described in detail. Some additional applications will
also be introduced.
Key words SPR, Protein, DNA, Reusable, Binding, Affinity, Kinetics
1 Introduction
SPR is an ideal technique for the study of protein–DNA interactions. It offers several advantages over more traditional in vitro
methods such as electrophoretic mobility shift assays or nitrocellulose filter binding assays. It can provide real-time monitoring of
binding and dissociation events, enabling the identification of binding and the measurement of affinity and kinetic parameters [1–3].
Furthermore, the technique is quick, sensitive, uses low amounts of
protein, and is highly automatable.
A traditional approach to study protein–DNA interactions by
SPR is to tether the DNA (the “ligand”) via a biotin tag to a
streptavidin (SA)-coated chip and then to flow the protein (the
“analyte”) over the top to monitor the interaction. However,
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
369