2-DIMENSIONAL ELECTROPHORESIS OF PROTEINS - AN UPDATED PROTOCOL AND IMPLICATIONS FOR A FUNCTIONAL-ANALYSIS OF THE GENOME

The two-dimensional electrophoresis (2-DE) technique developed by Klos e in 1975 (Humangenetik 1975, 26, 211-234), independently of the techn ique 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 sin ce its introduction. Many modifications and suggestions found in the l iterature were also tested and then integrated into our original metho d if advantageous. Gel and buffer composition, size of gels, use of st acking gels or not, necessity of isoelectric focusing (IEF) gel incuba tion, 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 cons titute our 2-DE method presented here, a few features are of particula r significance: (i) sample loading onto the acid side of the IEF gel w ith the result that both acidic and basic proteins are well resolved i n the same gel; (ii) use of large (46 X 30 cm) gels to achieve high re solution, but without the need of unusually large, fiat gel equipment; (iii) preparation of ready-made gel solutions which can be stored fro zen, 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 literatu re for 2-DE of complex protein mixtures. The 2-DE patterns were of hig h quality with regard to spot shape and background. The reproducibilit y of the protein patterns is demonstrated and shown to be thoroughly s atisfactory. An example is given to show how effectively 2-DE of high resolution and reproducibility can be used to study the genetic variab ility of proteins in an interspecific mouse backcross (Mus musculus X Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structu ral analysis of the human genome, currently pursued most intensively o n a worldwide scale, should be accompanied by a functional analysis of the genome Mat starts from the proteins of the organism.