E3 ligases remain among the most difficult enzyme targets to assay and discover potent therapeutic applications, possibly due to their dependence on upstream E1 and E2 enzymes for the activation and transfer of ubiquitin. As a result, traditional catalytic assays for E3 ligases are complex and prone to off-target false positives. At LifeSensors, we have developed reagents and assays encompassing both traditional and non-traditional approaches for E3 ligase screening and profiling. Quickly distinguishing true hits from false positives, developing structure-activity relationships, and establishing rank order potency from purified enzymes to cellular models are key steps to success. LifeSensors brings together the tools and expertise necessary to overcome the many pitfalls in the E3 ligase field.
Our ELISA-based assay is a proven, high throughput method for monitoring E3 ligase activity. It is a perfect low-cost option for small molecule screening. E3 ELISA assay is based on the capture of the polyubiquitinated E3 ligase or the E3’s substrate by a proprietary polyubiquitin binding reagent. Labelled Tandem Ubiquitin Binding Entities (TUBEs) are used to detect the polyubiquitination by chemiluminescence.