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.
