In Vitro Ubiquitination and E3 Ligase Activity Assays

A degrader works only if the ternary complex it forms is productive, meaning that the recruited E3 ligase transfers ubiquitin onto the target protein. Once the enzyme system has been assembled and qualified, the next question is whether a specific compound drives that transfer, and how efficiently it does so relative to other compounds in the series. Reconstituted in vitro reactions answer this question with defined components, ahead of cellular work.

Amerigo Scientific supplies assay kits that quantify PROTAC-mediated and molecular glue-mediated ubiquitination of a target of choice, customizable kits that measure E3 ligase activity for any E2/E3 pair or E3/substrate pair, and gel-based kits for direct visualization of ubiquitin conjugates.

PROTAC In Vitro Ubiquitination Assays

PROTACs are heterobifunctional small molecules that bind a target protein and a ubiquitin E3 ligase at the same time, which leads to ubiquitination and degradation of the target. The efficiency of a PROTAC therefore depends on the intrinsic ability of the target protein to be ubiquitinated once the ternary complex has formed, and this property can be measured directly in a reconstituted reaction.

We provide in vitro ubiquitination kits developed as a high throughput approach for predicting PROTAC efficiency, available for three E3 ubiquitin ligases: Cereblon, VHL and HDM2. A colorimetric version allows quantitative measurement on absorbance-based plate readers at 650 nm and 480 nm, with day-to-day consistency that exceeds luminescence-based detection, and assay plates are available separately for laboratories that supply their own reaction components.

Kit Technologies

The in vitro ubiquitination platform was designed to monitor multiple variants of PROTAC with variable linkerology, exit vectors, variable ligands for an E3 ligase, or targets to evaluate the best combination that results in robust ubiquitination.

Comparison

*half-maximal degradation concentration

Molecular Glue In Vitro Ubiquitination Assays

Molecular glues are naturally occurring molecules that bind an E3 ligase and a target protein, which leads to ubiquitination and degradation of the target. As with PROTACs, the efficiency of a molecular glue tracks the intrinsic ability of the target protein to be ubiquitinated, so the same reconstituted measurement applies.

Amerigo Scientific provides an in vitro ubiquitination kit developed as a high throughput approach for predicting molecular glue efficiency by monitoring ubiquitination of the target protein.

Customizable E3 Ligase Activity Assays

E3 ligases act as scaffolds that dock an E2 conjugating enzyme alongside a target substrate and mediate transfer of ubiquitin from the E2 thioester intermediate onto the substrate. E3 ligases also ubiquitinate themselves, and this autoubiquitination is used in vitro as a direct reporter of ligase activity. Quantifying the polyubiquitin produced in either reaction gives a direct measure of E3 ligase activity.

Our kits built on a plate pre-coated with a proprietary polyubiquitin capture reagent, where the product of the E3 ligase-mediated reaction is captured and quantified. The E3LITE format supports the focused investigation of any E2/E3 enzyme pair and of particular polyubiquitin linkage types, while the autoubiquitination and substrate ubiquitination format extends the same platform to any E3 and substrate pair. Both have been validated for high throughput screening and have been used to discover compounds that selectively modulate E3 ligase function. Chemiluminescent and colorimetric detection are both available, the latter reading at 650 nm and 480 nm on absorbance-based plate readers, and plates are supplied in modular 96-well and 384-well formats.

Gel-Based E3 Ligase Activity Assays

Ubiquitin is conjugated through its C-terminus to lysine side chains on a target protein, and further ubiquitin moieties can be added through any of the seven lysine residues of ubiquitin itself to build polyubiquitin chains, of which K48-linked chains are the best characterized and the most closely associated with proteasomal degradation. Because each added ubiquitin shifts the mass of the conjugate by roughly 8.6 kDa, gel separation resolves mono-ubiquitinated and polyubiquitinated species as a ladder and shows the extent of chain formation directly.

We provide customizable gel-based kits in which the reaction is run with the E1, E2 and E3 enzymes of choice and separated by SDS-PAGE. The Alexa647 format uses fluorescently labeled ubiquitin for direct visualization and quantification of the gel, and the VU1-HRP format detects conjugated ubiquitin with an anti-ubiquitin antibody. Both are supplied with assay buffer, E1, ubiquitin mix, ATP, a positive control enzyme pair and gel loading buffer, and both replace the longer western blot workflow conventionally used for E3 ligase assays.

Features

Both kits can be used as an orthogonal assay to confirm results from high throughput screening campaigns.