Extraction of Escherichia coil proteins with organic solvents prior to two-dimensional electrophoresis

M. P. Molloy, B. R. Herbert, K. L. Williams*, A. A. Gooley

*Corresponding author for this work

Research output: Contribution to journalArticle

99 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)701-704
Number of pages4
JournalElectrophoresis
Volume20
Issue number4-5
DOIs
Publication statusPublished - 1999
Externally publishedYes

Keywords

  • Extraction
  • Organic solvent
  • Proteome
  • Solubility
  • Two-dimensional electrophoresis

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