2 REPLICA BLOTTING METHODS FOR FAST IMMUNOLOGICAL ANALYSIS OF COMMON PROTEINS IN 2-DIMENSIONAL ELECTROPHORESIS

The combination of two-dimensional electrophoresis (2-DE) and subseque nt Western blot analysis with antibodies directed against common cellu lar proteins is a straightforward and reliable method to quickly gener ate fix points in a protein map. In order to assure high accuracy in t he allocation of protein spots, two different replica blotting methods for semidry blotting devices were established. The first of the two w as described by Johansson (Electrophoresis 1987, 8, 379-383). By syste matically changing the direction of the blotting current, proteins wer e 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 m ethod described here combines the standard blotting procedure with the generation of a 'contact copy' from the gel. Both systems offer the p ossibility to subject one membrane to antibody-mediated imaging, while the second membrane can be stained with highly sensitive total protei n detection procedures. Protein identification is then carried out by comparing the signals on both matrices.