High-Throughput Arginylation Assay in Microplate Format

Here, we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for the identification of small molecule inhibitors and activators of ATE1, high-volume analysis of AE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified 2 compounds which specifically affect ATE1-regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of beta-actin's N-terminal peptide, but it can also be applied using other ATE1 substrates.