Summary
Assaying sequence-specific DNA–protein complex formation in vitro often involves the use of specific labelling or modification of the components of the complex to provide unique signals that can be used to assess the affinity of the interaction. Surface plasmon resonance (SPR) spectroscopy is an optical technique that can be used without radio- or other labelling of the components of a complex provided that one of the partners can be immobilised to a solid support. For DNA oligonucleotides this can easily be achieved by the incorporation of a biotin end label, but proteins can also be immobilised if they carry conventional tags for affinity purification, such as GST or polyhistidine extensions. The SPR effect relies on changes in the refractive index of solutions adjacent to the immobilised surface and is extremely sensitive. The continuous flow systems developed by BIAcore AB (now GE Healthcare Biosciences AB) permit real-time recording of the binding and dissociation of analyte species to the immobilised ligand, resulting in both rapid stoichiometric kinetic, affinity, and thermodynamic measurements. These assays can be carried out with complex mixtures of analytes, providing a powerful addition to the techniques available to probe such interactions. We illustrate the use of such assays here using the example of the E. coli methionine repressor, MetJ, which is also described in Chapters “Filter-Binding Assays” and “Ethylation Interference Footprinting of DNA–Protein Complexes.”
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Persson, B., Buckle, M. M. and Stockley, P. G. (2000). Kinetics of DNA interactions studied by surface plasmon resonance spectroscopy. In DNA–Protein Interactions: A Practical Approach. (Editors, Travers, A. and Buckle, M. M.), Chapter 19, pp. 257–279, Oxford University Press, Oxford, UK.
Biacore web page (General Electric Company); http://www.biacore.com.
Rich, R. L. and Myzska, D. G. (2006). Survey of the year 2006 commercial optical biosensor literature. J. Mol. Recognit. 20, 300–366.
Buckle, M., Williams, R. M., Negroni, M., and Buc, H. (1996). Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance. Proc. Natl Acad. Sci. U. S. A. 93, 889–894.
Fisher, R. J. and Fivash, M. (1994). Surface plasmon resonance based methods for measuring the kinetics and binding affinities of biomolecular interactions. Curr. Opin. Biotechnol. 5, 389–395.
Di Primo, C. and Lebars, I. (2007) Determination of refractive index increment ratios for protein–nucleic acid complexes by surface plasmon resonance. Anal. Biochem. 368, 148–155.
Parsons, I. D. and Stockley, P. G. (1997) Quantitation of the E. coli methionine repressor–operator interaction by surface plasmon resonance is not affected by the presence of a dextran matrix. Anal. Biochem. 254, 82–87.
Somers, W. S. and Phillips, S. E. V. (1992). Crystal structure of the met repressor operator complex at 2.8 Ǻ resolution: DNA recognition by β strands. Nature 346, 586–590.
Phillips, S. E. V., Manfield, I., Parsons, I. D., Davidson, B., Rafferty, J. B, Somers, W. S., Cohen, G., Saint-Girons, I. and Stockley, P. G. (1989) Cooperative tandem binding of met repressor of Escherichia coli. Nature 341, 711–715.
Parsons, I. D., Persson, B., Mekhalfia, A., Blackburn, G. M., and Stockley, P. G. (1995). Probing the molecular mechanism of action of co-repressor in the Escherichia coli methionine repressor–operator complex using surface-plasmon resonance (SPR). Nucleic Acids Res. 23, 211–216.
Schuck, P. (1997). Reliable determination of binding affinity and kinetics using surface plasmon resonance biosensors. Curr. Opin. Biotechnol. 8, 498–502.
Schuck, P. and Minton, A. P. (1996). Kinetic analysis of biosensor data: elementary tests for self-consistency. Trends Biochem. Sci. 21, 458–460.
Schuck, P. and Minton, A. P. (1996) Analysis of mass transport-limited binding kinetics in evanescent wave biosensors. Anal. Biochem. 240, 262–272.
Schuck, P. (1996) Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport. Biophys. J. 70, 1230–1249.
Acknowledgements
We thank various colleagues for their help with the SPR experiments reported here, especially Dr Isobel D. Parsons. We also acknowledge previous discussions and interactions with Dr Malcolm Buckle, and Dr Francis Markey for valuable comments on the presentation of Biacore technology.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Stockley, P.G., Persson, B. (2009). Surface Plasmon Resonance Assays of DNA-Protein Interactions. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_38
Download citation
DOI: https://doi.org/10.1007/978-1-60327-015-1_38
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-014-4
Online ISBN: 978-1-60327-015-1
eBook Packages: Springer Protocols