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Extraction of Escherichia coli proteins with organic solvents prior to two‐dimensional electrophoresis
Author(s) -
Molloy Mark P.,
Herbert Ben R.,
Williams Keith L.,
Gooley Andrew A.
Publication year - 1999
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/(sici)1522-2683(19990101)20:4/5<701::aid-elps701>3.0.co;2-5
Subject(s) - chromatography , proteome , chemistry , chloroform , thiourea , extraction (chemistry) , urea , membrane protein , electrophoresis , peptide mass fingerprinting , protein purification , gel electrophoresis , biochemistry , membrane , proteomics , organic chemistry , gene
Compared to soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on two‐dimensional (2‐D) gels. One possibility is that many hydrophobic proteins are simply not extracted from the sample prior to 2‐D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solvents, then reconstituting the extracted proteins in highly solubilising sample solution amenable to 2‐D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform and methanol, followed by solubilisation using a combination of urea, thiourea, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprinting assisted in the identification of 13 proteins, 8 of which have not previously been reported on 2‐D gels. Five of these new proteins possess a positive hydropathy plot. These results suggest that organic solvent extractions may be useful for selectively isolating some proteins that have previously been missing from proteome maps.