If an antibody specific for the protein of interest is available, immunological detection and immunoelectrophoresis are the preferred methods for identification of that protein. Specifically, since the immuno-reactive site on a protein need not be affected by any post-translational modifications of a protein, immunological identification is the principal method for the recognition of a particular gene product in an electrophoretic pattern. Blotting of protein gel electrophoresis patterns to a membrane, with subsequent exposure of the blot to antibody, is the primary tool of immunological identification. More than any other factor, it is responsible for the possibility to construct protein databases from the hundreds to thousands of spots separable by two-dimensional gel electrophoresis .
Immunoelectrophoresis is the electrophoresis of one or more macromolecule in gels containing specific antibodies against them. Depending on the relative concentrations of the antibody and macromolecule, plus their affinity for each other, they interact to form complexes, which can then immunoprecipitate within the gel. The precipitates are visible within the gel. In this way, it is possible to quantify the amount of macromolecule present, by measurement of the size of the "rocket" produced and comparison with a known standard concentration (1). The antigenic relationships between proteins can be inferred from the precipitation patterns generated when the two are subjected to immunoelectrophoresis in adjacent wells of the gel, so that their precipitation patterns overlap (Fig. 1). If the overlapping rockets behave independently, the two molecules responsible are not immunologically related. If, however, they generate "fused rockets," at least some of the same antibody molecules recognize both macromolecules. Crossed immunoelectrophoresis (Fig. 1) consists of an initial gel electrophoresis separation of the macromolecules of interest, followed by their electrophoresis at a right angle to the initial separation into a gel containing the antibody; this technique can yield particularly high resolution (3).
Figure 1. Crossed immunoelectropherogram of human serum. First dimension: Electrophoresis occurred in 1% (w/v) agarose, sodium barbital buffer, pH 8.6, 0.02 Mionic strength, at 10 V/cm and 15°C, for 70 min, with the anode to the left. The circular well where the sample of human serum was applied is visible at the right. Second dimension: Electrophoresis occurred in the same gel and gel buffer, but containing 12.5 ^l rabbit antibody against human serum/cm2 of gel, at 2 V/cm, for 20 h, with the anode at the top. The antibody is isoelectric at pH 8.6 and therefore stationary during electrophoresis.
