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Isolation and preparation of bacterial cell walls for compositional analysis by Ultra Performance Liquid Chromatography
Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
2014 (Engelska)Ingår i: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, nr 83, s. UNSP e51183-Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

Ort, förlag, år, upplaga, sidor
JoVE , 2014. nr 83, s. UNSP e51183-
Nyckelord [en]
Chemistry, Issue 83, peptidoglycan, bacterial cell wall, ultra-performance liquid chromatography, gh-performance liquid chromatography, cell shape, morphogenesis, LAROSA EJ, 1985, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF
Nationell ämneskategori
Mikrobiologi inom det medicinska området Medicinsk laboratorie- och mätteknik
Identifikatorer
URN: urn:nbn:se:umu:diva-100312DOI: 10.3791/51183ISI: 000348513500058PubMedID: 24457605OAI: oai:DiVA.org:umu-100312DiVA, id: diva2:792194
Tillgänglig från: 2015-03-03 Skapad: 2015-02-27 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Cava, Felipe

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Molekylär Infektionsmedicin, Sverige (MIMS)Institutionen för molekylärbiologi (Medicinska fakulteten)
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Journal of Visualized Experiments
Mikrobiologi inom det medicinska områdetMedicinsk laboratorie- och mätteknik

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