Abstract

The two-dimensional electrophoretic O'Farrell technique can be simplified in terms of effort and equipment. This simplification derives mainly from the fact that the first dimension (isoelectric focusing) is run in a vertical slab gel. Since all tracks are cast in a single template, a reproducible and uniform first dimension is obtained, allowing easy and accurate comparison among tracks after standard staining and/or radioautographic procedures. After the first dimension, the focused tracks are cut and, following equilibration, simply laid on top of the second dimensional slab gel, whose spacers are 0.1 mm thicker than those used in the first separation. Thus, basically the same equipment can be used for both dimensions. This modified technique has been experimentally validated since it has allowed the detection of structural variants of two proteins related to the mouse complement system, namely, the fourth component and the sex-limited protein.