Abstract
Many single-molecule experimental techniques exploit fluorescence as a tool to investigate conformational dynamics and molecular interactions or track the movement of proteins in order to gain insight into their biological functions. A prerequisite to these experimental approaches is to graft one or more fluorophores on the protein of interest with the desired photophysical properties. Here, we present detailed procedures for the current most efficient methods used to covalently attach fluorophores to proteins. Alternative direct and indirect labeling strategies are also described.
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References
Nathan C. Shaner, Paul A. Steinbach, & Roger Y. Tsien. (2005). A guide to choosing fluorescent proteins. Nature Methods. 2, 905–909.
Petukhova GV, Pezza RJ, Vanevski F, Ploquin M, Masson JY, Camerini-Otero RD. (2005). The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination. Nat. Struct. Mol. Biol. 12, 449–453.
Modesti M, Ristic D, van der Heijden T, Dekker C, van Mameren J, Peterman EJ, Wuite GJ, Kanaar R, Wyman C. (2007). Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule. Structure. 15, 599–609.
Richard DJ, Bolderson E, Cubeddu L, Wadsworth RI, Savage K, Sharma GG, Nicolette ML, Tsvetanov S, McIlwraith MJ, Pandita RK, Takeda S, Hay RT, Gautier J, West SC, Paull TT, Pandita TK, White MF, Khanna KK. (2008). Single-stranded DNA-binding protein hSSB1 is critical for genomic stability. Nature. 453, 677–681.
Galletto R., Amitani I., Baskin R.J., Kowalczykowski S.C. (2006). Direct observation of individual RecA filaments assembling on single DNA molecules. Nature. 443, 875–878.
Henricksen LA, Umbricht CB, Wold MS. (1994) Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132.
Visnapuu M.-L., Duzdevich, D. and Greene, E. C. (2007). Using Total Internal Reflection Fluorescence Microscopy, DNA Curtains, and Quantum Dots to Investigate Protein-DNA Interactions at the Single-molecule Level. Modern Research and Educational Topics in Microscopy. Méndez-Vilas, A. and Diaz, J. (Eds). Microscopy series N° 3 Vol. 1, pp 297–308.
Visnapuu ML, Greene EC. (2009). Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nat. Struct. Mol. Biol. 16, 1056–1062.
Acknowledgments
Work in our laboratory is supported by Laserlab-Europe, the Association for International Cancer Research (AICR), the Agence pour la Recherche contre le Cancer (ARC), The city of Marseille, The Region of Provence-Alpes-Côtes d’Azur, and the Mediterranean Institute of Microbiology (IMM/IFR88). We would like to thank Sabrina Lignon from the mass spectrometry platform, Marielle Bauzan from the fermentation unit, and Yann Denis from the transcriptome platform of the IMM/IFR88 for advice and help with instrumentation and services. We thank Marc Wold (University of Iowa) for the gift of the p11d-tRPA polycistronic expression construct.
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Modesti, M. (2011). Fluorescent Labeling of Proteins. In: Peterman, E., Wuite, G. (eds) Single Molecule Analysis. Methods in Molecular Biology, vol 783. Humana Press. https://doi.org/10.1007/978-1-61779-282-3_6
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DOI: https://doi.org/10.1007/978-1-61779-282-3_6
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