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A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors

By Stephen K-F. Wong

Phosphorylation of extracellular signal-regulated kinase (ERK)is linked to activation of many cell surface receptors and kinases. However, phosphorylated ERK has not been used as a biochemical marker to monitor pharmacology of these biomolecules,largely because commonly used methods to detect the phosphoprotein are not quantitative and do not have sufficient throughput. In this article,a high-throughput, 384-well,cell-based functional assay to quantify dopamine agonist-induced ERK phosphorylation in D2- and D3-overexpressed cell lines is described.The assay uses infrared-labeled secondary antibodies to detect phospho-ERK,and the signals in the wells of the microtiter plate are quantified by a LI-COR infrared scanner.