High-throughput arginylation assay in microplate format

Abstract

Here we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for identifi cation of small-molecule inhibitors and activators of ATE1, high-volume analysis of ATE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identifi ed two compounds which specifi cally affect ATE1- regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of betaactin’s N-terminal peptide, but it can also be applied using other ATE1 substrates.