Detection of genipin/geniposide-target molecules by a geniposide overlay method using anti-geniposide antibody

A geniposide overlay method for the detection of genipin/geniposide-binding proteins was attempted using a specific anti-geniposide antiserum. The specific anti-geniposide antiserum that recognized the genipin moiety was obtained from rabbits immunized with geniposide-rabbit serum albumin (RSA) conjugate as antigen. The results of the overlay method revealed a major geniposide binding protein with a molecular weight about 170 kDa in the cytosolic fraction of rat brain cortex and PC12h cells. The major protein band was comparable with the protein band (about 170 kDa) detected by simultaneous Western blot analysis using anti-neuronal nitric oxide synthase (nNOS) antiserum. Furthermore, geniposide activated NOS activity in a concentration-dependent manner. These result; indicate that the antiserum obtained was useful for the detection of a genipin/geniposide-target molecule and strongly suggested that direct binding to and activating of nNOS cause the neuritogenic activity of genipin/geniposide in PC12h cells.