'Crossed immunoblotting': identification of proteins after crossed immunoelectrophoresis and electrotransfer to nitrocellulose membranes.

Human serum proteins or membrane proteins from human erythrocytes were separated by crossed immunoelectrophoresis in agarose gels under non-denaturating and non-reducing conditions and precipitated by polyspecific antibodies against the respective protein mixtures. The separated proteins were subsequently transferred electrophoretically to nitrocellulose sheets and incubated with mouse monoclonal antibodies against individual protein species. Visualization of the complexes of antigen and monoclonal antibody on the nitrocellulose sheets was performed with peroxidase-conjugated second antibodies. The additional electroblotting step clearly improved the analytical resolution possibilities of crossed immunoelectrophoresis: crossed immunoblotting allows a straightforward identification of individual proteins in complex crossed immunoelectrophoresis patterns. At the same time separation and characterization of proteins is performed under non-denaturing conditions, which is not possible with blotting techniques based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.