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
- Publication Type:
- Journal Article
- Citation:
- Electrophoresis, 2006, 27 (8), pp. 1630 - 1640
- Issue Date:
- 2006-04-01
Closed Access
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2006004003.pdf | 1.05 MB |
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Full metadata record
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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