Chapter title |
Effector Translocation Assay: Differential Solubilization
|
---|---|
Chapter number | 35 |
Book title |
Bacterial Protein Secretion Systems
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-7033-9_35 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7031-5, 978-1-4939-7033-9
|
Authors |
Irina S. Franco, Sara V. Pais, Nuno Charro, Luís Jaime Mota |
Editors |
Laure Journet, Eric Cascales |
Abstract |
The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringelike nanomachines is an important step toward understanding the mechanisms underlying the virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a nonionic detergent (Triton X-100) enables solubilization of host cell membranes but not of bacterial membranes. This allows the isolation of a Triton-soluble fraction lacking bacteria but enriched in proteins present in the host cell cytoplasm and plasma membrane. Using appropriate controls, this fraction can be probed by immunoblotting for the presence of bacterial effector proteins delivered into host cells. |
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