Sample prefractionation in granulated sephadex IEF gels.

Abstract

Prefractionation procedures not only aid in reducing sample complexity, but also permit loading of higher protein amounts within the separation range applied to two-dimensional electrophoresis (2-DE) gels and thus facilitate the detection of less abundant protein species. Hence we developed a simple, cheap, and fast prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated Sephadex gels, containing chaotropes, zwitterionic detergents and carrier ampholytes. After IEF, up to ten Sephadex gel fractions alongside the pH gradient are obtained, and then applied directly onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of 2-DE. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and full compatibility with subsequent 2-DE, because the prefractionated samples have not to be eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with IEF in IPG strips. This sample prefractionation method has been successfully applied for the separation, detection and identification of low abundance proteins from pro- and eukaryotic samples.