Abstract
Protein function is generally dependent on its subcellular localisation. In Gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different compartments: the cytoplasm, the inner membrane, the periplasm, the outer membrane and the extracellular medium. Different approaches can be used to determine the protein localisation within a cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labelling, optical fluorescence microscopy, and biochemical technics. In this chapter, we describe a simple and efficient method to isolate the different compartments of Escherichia coli by a fractionation method and to determine the presence of the protein of interest. For inner membrane proteins we propose a method to discriminate between integral and peripheral membrane proteins.
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Acknowledgments
This work was supported by the Centre National de la Recherche Scientifique and the Agence National de la Recherche (ANR-14-CE09-0023).
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Petiti, M., Houot, L., Duché, D. (2017). Cell Fractionation. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_3
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DOI: https://doi.org/10.1007/978-1-4939-7033-9_3
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