Improved 2-DE of microorganisms after acidic extraction

Herbert, Ben R., Grinyer, Jasmine, McCarthy, John T., Isaacs, Mitchell, Harry, Elizabeth J., Nevalainen, Helena, Traini, Mathew D., Hunt, Sybille, Schulz, Ben, Laver, Matthew, Goodall, Anthony R., Packer, Joanne, Harry, Jenny L. and Williams, Keith L. (2006) Improved 2-DE of microorganisms after acidic extraction. Electrophoresis, 27 8: 1630-1640. doi:10.1002/elps.200500753


Author Herbert, Ben R.
Grinyer, Jasmine
McCarthy, John T.
Isaacs, Mitchell
Harry, Elizabeth J.
Nevalainen, Helena
Traini, Mathew D.
Hunt, Sybille
Schulz, Ben
Laver, Matthew
Goodall, Anthony R.
Packer, Joanne
Harry, Jenny L.
Williams, Keith L.
Title Improved 2-DE of microorganisms after acidic extraction
Journal name Electrophoresis   Check publisher's open access policy
ISSN 1522-2683
0173-0835
Publication date 2006-04
Sub-type Article (original research)
DOI 10.1002/elps.200500753
Volume 27
Issue 8
Start page 1630
End page 1640
Total pages 11
Place of publication Weinheim
Publisher Verlag Chemie, GmbH.
Language eng
Subject 03 Chemical Sciences
0301 Analytical Chemistry
06 Biological Sciences
0601 Biochemistry and Cell Biology
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.
Keyword Proteomics
Cell wall
Acidic extraction
2-DE
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Chemistry and Molecular Biosciences
 
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Created: Fri, 18 Dec 2009, 11:44:31 EST by Ms May Balasaize on behalf of Faculty of Science