A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer

Posttranslational modification of protein by lysine-48 (K48) linked ubiquitin (Ub) chains is the major cellular mechanism for selective protein degradation that critically impacts biological processes such as cell cycle checkpoints. In this chapter, we describe an in vitro biochemical approach to detect a K48-linked di-Ub chain by fluorescence resonance energy transfer (FRET). To this end, we detail methods for the preparation of the relevant enzymes and substrates, as well as for the execution of the reaction with high efficiency. Tracking K48 polyubiquitination using this sensitive and highly reproducible format provides an opportunity for high-throughput screening that leads to identification of small molecule modulators capable of changing ubiquitination for improving human health.