Preparation of dopamine transporter-specific antibodies using molecular cloned genes

Dopamine transporter (DAT) plays the most important role in terminating the actions of dopamines released into the synaptic cleft. DAT is also the target of various psychotropic drugs such as cocaine and amphetamine. In this study we prepared DAT-specific antibodies using the 2nd extracellular loop of rat DAT as an antigen. The 2nd extracellular loop of the rat DAT was expressed in bacteria as a fusion protein with glutathione-S-transferase, and injected into rabbits to raise antibodies. Produced antibodies clearly recognized the rat DAT in ELISA, immunoblotting, and immunoprecipitation. As expected from the high sequence homology between the rat and human DAT, the antibodies raised for the rat DAT cross-reacted with the human DAT in the immunoblotting. Considering the specificity for DAT with wide range of applications such as ELISA, immunoblotting, and immunoprecipitation, these antibodies would be valuable tool for understanding the pharmacological actions of dopamine transporter and drug addiction.