Two replica blotting methods for fast immunological analysis of common proteins in two-dimensional electrophoresis

The combination of two-dimensional electrophoresis (2-DE) and subsequent Western blot analysis with antibodies directed against common cellular proteins is a straightforward and reliable method to quickly generate fix points in a protein map. In order to assure high accuracy in the allocation of protein spots, two different replica blotting methods for semidry blotting devices were established. The first of the two was described by Johansson (Electrophoresis 1987, 8, 379-383). By systematically changing the direction of the blotting current, proteins were simultaneously transferred from one gel onto two membranes placed at both sides of the gel. However, several modifications of this method were necessary in order to use a semidry blotting device. The second method described here combines the standard blotting procedure with the generation of a 'contact copy' from the gel. Both systems offer the possibility to subject one membrane to antibody-mediated imaging, while the second membrane can be stained with highly sensitive total protein detection procedures. Protein identification is then carried out by comparing the signals on both matrices.