Effector translocation assay

Differential solubilization

Irina S. Franco, Sara V. Pais, Nuno Charro, Luís Jaime Mota

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages501-515
Number of pages15
Volume1615
DOIs
Publication statusPublished - 1 Jan 2017

Publication series

NameMethods in Molecular Biology
Volume1615
ISSN (Print)1064-3745

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Keywords

  • Bacterial protein secretion system
  • Detergent solubilization
  • Effector
  • Immunoblotting
  • SDS-PAGE
  • Translocation
  • Type III secretion

Cite this

Franco, I. S., Pais, S. V., Charro, N., & Mota, L. J. (2017). Effector translocation assay: Differential solubilization. In Methods in Molecular Biology (Vol. 1615, pp. 501-515). (Methods in Molecular Biology; Vol. 1615). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7033-9_35