Abstract
Surface plasmon resonance (SPR) is a widely employed technique for studying protein–protein interactions. Here, we describe a method for the analysis of single-stranded DNA binding protein (SSB)–heterologous protein interactions by SPR. This method avoids several pitfalls often associated with SPR, particularly difficulties in immobilizing the protein while still allowing for facile regeneration of the sensor chip surface for subsequent experiments. Essentially, the method entails immobilizing a biotinylated single-stranded DNA oligo onto the chip surface, which is then bound by SSB prior to analyte addition to the SSB-coated chip. This allows for rapid qualitative and detailed quantitative analysis of both equilibrium and kinetic parameters of the SSB–protein interaction.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jonsson U, Fagerstam L, Ivarsson B, Johnsson B, Karlsson R, Lundh K, Lofas S, Persson B, Roos H, Ronnberg I, Sjolander S, Stenberg E, Stahlberg R, Urbaniczky C, Ostlin H, Malmqvist M (1991) Real-time biospecific interaction analysis using surface-plasmon resonance and a sensor chip technology. BioTechniques 11:620–627
Fagerstam LG, Frostell-Karlsson A, Karlsson R, Persson B, Ronnberg I (1992) Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis. J Chromatogr 597:397–410
Witte G, Urbanke C, Curth U (2003) DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery. Nucleic Acids Res 31:4434–4440
Purnapatre K, Handa P, Venkatesh J, Varshney U (1999) Differential effects of single-stranded DNA binding proteins (SSBs) on uracil DNA glycosylases (UDGs) from Escherichia coli and mycobacteria. Nucleic Acids Res 27:3487–3492
Page AN, George NP, Marceau AH, Cox MM, Keck JL (2011) Structure and biochemical activities of Escherichia coli MgsA. J Biol Chem. 286:12075–12085.
Kelman Z, Yuzhakov A, Andjelkovic J, O'Donnell M (1998) Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly. EMBO J 17:2436–2449
Nicholson MW, Barclay AN, Singer MS, Rosen SD, van der Merwe PA (1998) Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1. J Biol Chem 273:763–770
Ehn M, Nilsson P, Uhlen M, Hober S (2001) Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein. Protein Expression Purif 22:120–127
Scaltriti E, Tegoni M, Rivetti C, Launay H, Masson JY, Magadan AH, Tremblay D, Moineau S, Ramoni R, Lichiere J, Campanacci V, Cambillau C, Ortiz-Lombardia M (2009) Structure and function of phage p2 ORF34(p2), a new type of single-stranded DNA binding protein. Mol Microbiol 73:1156–1170
Morton TA, Myszka DG, Chaiken IM (1995) Interpreting complex binding kinetics from optical biosensors: a comparison of analysis by linearization, the integrated rate equation, and numerical integration. Anal Biochem 227:176–185
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Page, A.N., George, N.P. (2012). Methods for Analysis of SSB–Protein Interactions by SPR. In: Keck, J. (eds) Single-Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 922. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-032-8_12
Download citation
DOI: https://doi.org/10.1007/978-1-62703-032-8_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-031-1
Online ISBN: 978-1-62703-032-8
eBook Packages: Springer Protocols