Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Matthew R. Richardson, Sean Liu, Heather N. Ringham, Victor Chan, Frank A. Witzmann

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM® IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that OFF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.

Original languageEnglish (US)
Pages (from-to)2637-2644
Number of pages8
JournalELECTROPHORESIS
Volume29
Issue number12
DOIs
StatePublished - Jun 1 2008

Keywords

  • 2-DE
  • Escherichia coli
  • Narrow pH range
  • Prefractionation
  • Solution isoelectric focusing

ASJC Scopus subject areas

  • Clinical Biochemistry

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