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

Fingerprint

Cell Membrane
Bacterial Proteins
Octoxynol
Immunoblotting
Detergents
Virulence
Cytoplasm
Proteins
Bacteria
Membranes
Infection

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
Franco, Irina S. ; Pais, Sara V. ; Charro, Nuno ; Mota, Luís Jaime. / Effector translocation assay : Differential solubilization. Methods in Molecular Biology. Vol. 1615 Humana Press Inc., 2017. pp. 501-515 (Methods in Molecular Biology).
@inbook{028d98d9563547b8ac7c8fbdc2a43c48,
title = "Effector translocation assay: Differential solubilization",
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.",
keywords = "Bacterial protein secretion system, Detergent solubilization, Effector, Immunoblotting, SDS-PAGE, Translocation, Type III secretion",
author = "Franco, {Irina S.} and Pais, {Sara V.} and Nuno Charro and Mota, {Lu{\'i}s Jaime}",
note = "sem pdf conforme despacho.",
year = "2017",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-7033-9_35",
language = "English",
volume = "1615",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "501--515",
booktitle = "Methods in Molecular Biology",

}

Franco, IS, Pais, SV, Charro, N & Mota, LJ 2017, Effector translocation assay: Differential solubilization. in Methods in Molecular Biology. vol. 1615, Methods in Molecular Biology, vol. 1615, Humana Press Inc., pp. 501-515. https://doi.org/10.1007/978-1-4939-7033-9_35

Effector translocation assay : Differential solubilization. / Franco, Irina S.; Pais, Sara V.; Charro, Nuno; Mota, Luís Jaime.

Methods in Molecular Biology. Vol. 1615 Humana Press Inc., 2017. p. 501-515 (Methods in Molecular Biology; Vol. 1615).

Research output: Chapter in Book/Report/Conference proceedingChapter

TY - CHAP

T1 - Effector translocation assay

T2 - Differential solubilization

AU - Franco, Irina S.

AU - Pais, Sara V.

AU - Charro, Nuno

AU - Mota, Luís Jaime

N1 - sem pdf conforme despacho.

PY - 2017/1/1

Y1 - 2017/1/1

N2 - 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.

AB - 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.

KW - Bacterial protein secretion system

KW - Detergent solubilization

KW - Effector

KW - Immunoblotting

KW - SDS-PAGE

KW - Translocation

KW - Type III secretion

UR - http://www.scopus.com/inward/record.url?scp=85021706602&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-7033-9_35

DO - 10.1007/978-1-4939-7033-9_35

M3 - Chapter

VL - 1615

T3 - Methods in Molecular Biology

SP - 501

EP - 515

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

Franco IS, Pais SV, Charro N, Mota LJ. Effector translocation assay: Differential solubilization. In Methods in Molecular Biology. Vol. 1615. Humana Press Inc. 2017. p. 501-515. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7033-9_35