We describe here that a simple diffusion blotting method can couple immunoblotting analysis with another biochemical technique in a single polyacrylamide gel. The efficiency of protein transfer was evaluated by serial dilutions of nephrosin, a metalloproteinase of the astacin family, and by immunodetection. It is estimated that diffusion blotting produces 25-50% of the signal intensity compared to the classical electrophoretic transfer method. However, with diffusion blotting it is possible to generate several replicas from a single gel. In addition, a protein blot can be obtained from a sodium dodecyl sulfate (SDS)-polyacrylamide gel for zymography assay or from a native polyacrylamide gel for electrophoretic mobility shift assay (EMSA). In this regard, a particular signal in zymography or EMSA can be confirmed by simultaneous immunoblotting analysis with a corresponding antiserum. Therefore, diffusion blotting allows a direct comparison of signals between gels and replicas in zymography assay and EMSA. These advantages make diffusion blotting desirable when partial loss of transfer efficiency can be tolerated or be compensated by a more sensitive immunodetection reaction using enhanced chemiluminescence substrates.