Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia coli.
Bacterial Outer Membrane Proteins
/ metabolism
Biological Transport
Cell Membrane
/ metabolism
Centrifugation, Density Gradient
Escherichia coli
/ metabolism
Escherichia coli Proteins
/ metabolism
Glycolipids
/ metabolism
Lipopolysaccharides
/ chemistry
Peptidoglycan
/ metabolism
Sucrose
/ metabolism
LPS biogenesis
Membrane fractionation
Sucrose gradient
Tricine-SDS-PAGE
Ultracentrifugation
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
23
9
2022
pubmed:
24
9
2022
medline:
28
9
2022
Statut:
ppublish
Résumé
Gram-negative diderm bacteria are characterized by a tripartite cell envelope, composed of an inner membrane (IM) and a lipopolysaccharide (LPS)-containing outer membrane (OM), separated by an aqueous space where the peptidoglycan is embedded. LPS is a peculiar glycolipid endowed with several biological activities. The biosynthesis and transport of LPS to its final location take place in every compartment of the cell envelope. Proteins and protein machineries with different subcellular localization are involved in this process to facilitate the trafficking of LPS across subcellular compartments that differ in their physicochemical proprieties. The fractionation of bacterial cell envelopes can give information on the status of the LPS biogenesis by allowing the analysis of LPS profiles and of the localization of proteins involved in the transport. Here, we describe a standardized protocol for membrane fractionation in Escherichia coli using sucrose density gradient centrifugation that separates the IM from the OM cellular fractions. Bacterial cells are first converted into spheroplasts and lysed; then the membrane fractions are collected by ultracentrifugation and separated at high speed by exploiting the differences in membrane density. The fractions obtained are analyzed for LPS total amount and electrophoretic profile.
Identifiants
pubmed: 36151491
doi: 10.1007/978-1-0716-2581-1_4
doi:
Substances chimiques
Bacterial Outer Membrane Proteins
0
Escherichia coli Proteins
0
Glycolipids
0
Lipopolysaccharides
0
Peptidoglycan
0
Sucrose
57-50-1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
53-69Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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