Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome

Abstract

The two‐dimensional electrophoresis (2‐DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211–234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007–4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels – these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2‐DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 × 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready‐made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2‐DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2‐DE of complex protein mixtures. The 2‐DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2‐DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus × Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.