The substrate and E3 substrate finder service leverages a robust, quantitative assay platform to measure E3 ubiquitin ligase activity and define its role in substrate ubiquitination, or conversely identify novel E3 ligases that target an important substrate.

The assay uses LifeSensors’ proprietary polyubiquitin capture reagents to sensitively detect ubiquitination events, enabling both broad evaluation of E3 activity in the presence of candidate substrates and focused interrogation of defined E3–substrate pairs. By selectively capturing polyubiquitinated species, the assay provides a direct readout of E3-dependent ubiquitin transfer under controlled conditions.

Detection is achieved through a chemiluminescent readout, providing superior sensitivity and dynamic range compared with traditional gel-based assays. The assay is formatted in a 96-well plate, allowing efficient screening of multiple substrates, conditions, or E3 variants in parallel, and enabling quantitative comparison across experimental variables. This plate-based format supports higher throughput and reproducibility while maintaining the mechanistic insight of biochemical ubiquitination assays.

A high-quality antibody is required against the target substrate to enable specific detection. In cases where a suitable antibody is unavailable, substrate identification and validation can instead be performed using the LifeSensors E3 ID service, which employs mass spectrometry–based proteomics to identify ubiquitinated substrates in an unbiased manner. Together, these complementary platforms provide a flexible and scalable approach to E3 substrate discovery and validation, tailored to the biological question and available reagents.