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Improved 2‐DE of microorganisms after acidic extraction
Author(s) -
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.
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500753
Subject(s) - microorganism , bacillus subtilis , extraction (chemistry) , yeast , cell disruption , cell wall , bacteria , citric acid , chromatography , chemistry , microbiology and biotechnology , biochemistry , saccharomyces cerevisiae , biology , escherichia coli , genetics , gene
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.