Isoelectric focusing and two-dimensional gel electrophoresis.
By far the highest resolution of all separation techniques for intact proteins in a single analytical run continues to be by the combination of isoelectric focusing (IEF) and SDS-PAGE, originally introduced by O'Farrell [(1975). J. Biol. Chem.250, 4007-4021]. This analytical platform has seen a number of significant advances and applications over the past 25years, including reproducibility using immobilized pH gradient (IPG) strips [Bjellqvist et al. (1982). J. Biochem. Biophys. Methods6, 317-339.], resolution in alkaline IEF using hydroxyethyldisulfide (HED) [Olsson et al. (2002). Proteomics2, 1630-1632], and quantification for differential expression proteomics on intact proteins on a global scale [DIGE; Unlu et al. (1997). Electrophoresis18, 2071-2077]. These major improvements will be highlighted in this chapter alongside the principle and theory of 2D gel electrophoresis, as well as detailed methods for general 2D gel electrophoresis best use protocols.
Friedman
David B
DB
Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee, USA.
Hoving
Sjouke
S
Westermeier
Reiner
R
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry / Q000600:supply & distribution; D007525:Isoelectric Focusing / Q000379:methods; D008954:Models, Biological; D011506:Proteins / Q000032:analysis / Q000737:chemistry
D007202:Indicators and Reagents; D011506:Proteins