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 tar get of various psychotropic drugs such as cocaine and amphetamine. In this study we prepared DAT-specific antibodies using the 2nd extracellular loop of rat BAT as an antigen. The 2nd extracellular loop of the rat DAT was exp ressed in bacteria as a fusion protein with glutathione-S-transferase, and injected into rabbits to raise antibodies. Produced antibodies clearly reco gnized the rat DAT in ELISA, immunoblotting, and immunoprecipitation. As ex pected from the high sequence homology between the rat and human DAT, the a ntibodies raised for the rat DAT crossreacted with the human DAT in the imm unoblotting. Considering the specificity for DAT with wide range of applica tions such as ELISA, immunoblotting, and immunoprecipitation, these antibod ies would be valuable tool for understanding the pharmacological actions of dopamine transporter and drug addiction.