Improved 2-DE of microorganisms after acidic extraction

Herbert, BR; Grinyer, J; McCarthy, JT; Isaacs, M; Harry, EJ; Nevalainen, H; Traini, MD; Hunt, S; Schulz, B; Laver, M; Goodall, AR; Packer, J; Harry, JL; Williams, KL

Improved 2-DE of microorganisms after acidic extraction

Herbert, BR

Grinyer, J McCarthy, JT Isaacs, M Harry, EJ

Nevalainen, H Traini, MD Hunt, S Schulz, B Laver, M Goodall, AR Packer, J Harry, JL Williams, KL

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Publication Type:: Journal Article
Citation:: Electrophoresis, 2006, 27 (8), pp. 1630 - 1640
Issue Date:: 2006-04-01

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Field	Value	Language
dc.contributor.author	Herbert, BR https://orcid.org/0000-0003-4697-8197	en_US
dc.contributor.author	Grinyer, J	en_US
dc.contributor.author	McCarthy, JT	en_US
dc.contributor.author	Isaacs, M	en_US
dc.contributor.author	Harry, EJ https://orcid.org/0000-0003-0858-9473	en_US
dc.contributor.author	Nevalainen, H	en_US
dc.contributor.author	Traini, MD	en_US
dc.contributor.author	Hunt, S	en_US
dc.contributor.author	Schulz, B	en_US
dc.contributor.author	Laver, M	en_US
dc.contributor.author	Goodall, AR	en_US
dc.contributor.author	Packer, J	en_US
dc.contributor.author	Harry, JL	en_US
dc.contributor.author	Williams, KL	en_US
dc.date.issued	2006-04-01	en_US
dc.identifier.citation	Electrophoresis, 2006, 27 (8), pp. 1630 - 1640	en_US
dc.identifier.issn	0173-0835	en_US
dc.identifier.uri	http://hdl.handle.net/10453/548
dc.description.abstract	2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100 kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH 3 by 80 mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.	en_US
dc.relation.ispartof	Electrophoresis	en_US
dc.relation.isbasedon	10.1002/elps.200500753	en_US
dc.subject.classification	Analytical Chemistry	en_US
dc.subject.mesh	Cell Wall	en_US
dc.subject.mesh	Bacillus subtilis	en_US
dc.subject.mesh	Escherichia coli	en_US
dc.subject.mesh	Saccharomyces cerevisiae	en_US
dc.subject.mesh	Trichoderma	en_US
dc.subject.mesh	Acids	en_US
dc.subject.mesh	Phosphoric Acids	en_US
dc.subject.mesh	Ascorbic Acid	en_US
dc.subject.mesh	Citric Acid	en_US
dc.subject.mesh	Artifacts	en_US
dc.subject.mesh	Electrophoresis, Gel, Two-Dimensional	en_US
dc.subject.mesh	Hydrogen-Ion Concentration	en_US
dc.subject.mesh	Mass Spectrometry	en_US
dc.title	Improved 2-DE of microorganisms after acidic extraction	en_US
dc.type	Journal Article
utslib.citation.volume	8	en_US
utslib.citation.volume	27	en_US
utslib.for	0301 Analytical Chemistry	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0904 Chemical Engineering	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	closed_access
pubs.issue	8	en_US
pubs.publication-status	Published	en_US
pubs.volume	27	en_US

Abstract:

2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100 kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH 3 by 80 mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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http://hdl.handle.net/10453/548