Abstract
We describe a procedure to detect protein binding to SSB by polyacrylamide gel electrophoresis under non-denaturing conditions. As an example, we show the interaction of Thermus thermophilus (Tth) SSB with its cognate RecO protein. The interaction is detected as decay of the band corresponding to SSB by addition of RecO. We also demonstrate analysis of the RecO–RecR interaction as another example of this method.
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
Costes A, Lecointe F, McGovern S, Quevillon-Cheruel S, Polard P (2010) The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks. PLoS Genet 6:e1001238
Inoue J, Nagae T, Mishima M, Ito Y, Shibata T, Mikawa T (2011) A mechanism for single-stranded DNA-binding protein (SSB) displacement from sindle-stranded DNA upon SSB-RecO interaction. J Biol Chem 286:6720–6732
Wittig I, Schagger H (2005) Advantages and limitations of clear-native PAGE. Proteomics 5:4338–4346
Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Inoue J, Shigemori Y, Mikawa T (2006) Improvement of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein. Nucleic Acids Res 34:e69
Inoue J, Honda M, Ikawa S, Shibata T, Mikawa T (2008) The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins. Nucleic Acids Res 36:94–109
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
Inoue, J., Mikawa, T. (2012). Use of Native Gels to Measure Protein Binding to SSB. 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_13
Download citation
DOI: https://doi.org/10.1007/978-1-62703-032-8_13
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