Improved 2-DE of microorganisms after acidic extraction.
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.
Herbert
Ben R
BR
Proteome Systems, North Ryde, Sydney, NSW, Australia. ben.herbert@uts.edu.au
Grinyer
Jasmine
J
McCarthy
John T
JT
Isaacs
Mitchell
M
Harry
Elizabeth J
EJ
Nevalainen
Helena
H
Traini
Mathew D
MD
Hunt
Sybille
S
Schulz
Ben
B
Laver
Matthew
M
Goodall
Anthony R
AR
Packer
Joanne
J
Harry
Jenny L
JL
Williams
Keith L
KL
D000143:Acids; D016477:Artifacts; D001205:Ascorbic Acid; D001412:Bacillus subtilis / Q000737:chemistry / Q000302:isolation & purification*; D002473:Cell Wall / Q000737:chemistry; D019343:Citric Acid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010756:Phosphoric Acids; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000302:isolation & purification*; D014242:Trichoderma / Q000737:chemistry / Q000302:isolation & purification*
D000143:Acids; D010756:Phosphoric Acids; D019343:Citric Acid; C030242:phosphoric acid; D001205:Ascorbic Acid