ChoosE2-Opti™ Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to identify E2 enzyme(s) that are functional partners for a purified E3 ubiquitin ligase of interest and lead to its self-polyubiquitination. The assay kit contains five E2 enzymes to be tested with the E3 ligase of interest, and two E3 ligases (SMURF1 and MDM2) to use as internal controls. It also contains an E1 enzyme, ATP, an optimized TRF Ubiquitin Mix, and a universal buffer.

It utilizes a Europium-labeled Ubiquitin Donor as well as Cy5-labeled Ubiquitin Acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into polyubiquitin chains, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses.

Figure 1: ChoosE2-Opti intrachain TR-FRET Assay Kit schematic