E3 Ligase Screening & Profiling


Hundreds of E3 ligases have been identified in mammalian ubiquitylation cascades. E3 ligases conjugate monoubiquitin or polyubiquitin with various chain architecture to protein substrates or themselves.

E3 ligases remain among the most difficult enzyme targets to assay, possibly due to their dependence on upstream E1 and E2 enzymes for the activation and transfer of ubiquitin. As a result, 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. LifeSensors’ small molecule library contains a collection of ligase-centric compounds, which can be employed as a gold standard for your ligase drug discovery efforts. Further, LifeSensors has the tools to quickly exclude off-target hits through a variety of complementary biochemical and biophysical validation assays. In E3 ligase drug discovery, connecting the dots is particularly important. 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.

Lifesensors has developed recombinant enzymes for studying E3 ligases including a variety of E1s, E2s, and E3s.  In addition, we have developed ubiquitin mutants to help you identify the type of chains generated by your E3 ligase.


E2 Identification

These quantitative, plate-based assays are used to determine the most optimal E2 for the E3 of interest. The E2 can determine the ubiquitin chain-linkage type produced and the ability of your E3 to ubiquitylate substrates. Thus, E2 selection is a vital part for optimal E3 ligase activity.

Polyubiquitin Chain Identification

Different types of ubiquitylation linkage patterns can have different functional consequences for the substrate. Knowing the type of chain and branching can give insights into the signaling mechanism and the role of your E3 or substrate of interest. LifeSensors’ E3 ligase assays represent a very effective method for polyubiquitin chain identification by monitoring the activity of the E3 ligase with WT- and Lysine- mutant ubiquitin.

E3 Ligase Screening and Compound Profiling

Small molecule and PROTAC libraries can be screened using LifeSensors’ E3 ligase assays. These assays can also be used to screen for multiple E3 ligases at the same time, thus establishing compound selectivity.


E3 ELISA assay

Our ELISA-based assay is a proven, high throughput method for monitoring E3 ligase activity. It is a perfect option for small molecule screening.

TR-FRET E3 assay

This detection system utilizes our TUBE technology to monitor E3 ligase activity by fluorescence signal. This high-throughput system has the capability to screen compound libraries for E3 ligase interaction.


A presentation of our E3 Ligase Screening and Profiling Platform is here.


Click on examples below