High-throughput assay to monitor formation of the E2-ubiquitin thioester intermediate

Targeting components of ubiquitination pathways for drug discovery necessitates the development of high-capacity assays that monitor the ubiquitination process at defined steps of the E1-E2-E3 cascade. This chapter describes the development of an assay based on time-resolved fluorescence to monitor formation of the thioester intermediate between ubiquitin-conjugating enzymes (E2s) and ubiquitin. The methodology is exemplified by an assay tailored for the ubiquitin-conjugating enzyme Cdc34. This assay setup can be easily adapted to other E2s and is suitable to screen small molecule inhibitors of E2-thioester formation in a high-throughput mode.