Purification of D 2 dopamine receptor by photoaffinity labelling, high-performance liquid chromatography and preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis

Abstract

[ 125I]N-azidophenethylspiperone ([ 125I]azido-NAPS) was used as a photoaffinity ligand for bovine D 2 dopamine receptor. On photolysis, [ 125I]azido-NAPS was covalently incorporated into a major band of 94 kDa in bovine striatal membrane as assessed by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide gel). The labelled D 2 receptor protein from striatal membrane was solubilized and subjected to HPLC using gel filtration (TSK G3000 SW) and hydroxyapatite gel (Pentax SH2010C), followed by two steps of preparative SDS-PAGE. The D 2 receptor protein could be obtained as a single major polypeptide on SDS-PAGE by either silver staining or autoradiography.