Knowing the kinetics of target protein ubiquitination is crucial for the development of protein degrader compounds like PROTACs or molecular glues. NanoBRET® Ubiquitination Assays and biochemical Lumit® Immunoassays are efficient methods for monitoring target protein ubiquitination.

View this application note to learn more about antibody-based approaches to study target protein ubiquitination.

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Does My Target Become Ubiquitinated?

Cell-Based Approaches

NanoBRET® technology can be used to measure the kinetics of target protein ubiquitination or, in an endpoint format, for applications such as measuring compound dose-response curves.

In NanoBRET® ubiquitination assays, the target protein serves as the energy donor and is expressed in cells as an exogenous transient NanoLuc® fusion, or an endogenously tagged HiBiT fusion in LgBiT-expressing cells. The HaloTag-Ub fusion is exogenously expressed as the energy acceptor. Live-cell NanoBRET® Assays are performed in real time using either endpoint or kinetic analysis. Similar to ternary complex formation, changes in ubiquitination are typically observed within 1–4 hours after compound addition.

Schematic illustration of a NanoBRET-ubiquitin complex.

Schematic illustration of a NanoBRET-ubiquitin complex.

brd4 ubiquitination kinetic monitoring

Kinetic monitoring of BRD4 ubiquitination. HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line using CRISPR/Cas9 gene editing. A stable clone was transfected with HaloTag®-Ubiquitin acceptor plasmid. Cells were treated with 1μM MZ1 or 1μM dBET1, and the NanoBRET® Nano-Glo® Kinetic Detection Reagent was used to measure target protein ubiquitination over time.

brd4 ubiquitination endpoint monitoring

BRD4 ubiquitination following PROTAC treatment using a live-cell endpoint assay. HEK293 cells were transfected with NanoLuc®-BRD4 and HaloTag®-Ubiquitin plasmids at a 1:100 donor:acceptor ratio, plated in the presence of HaloTag® NanoBRET® 618 Ligand, and treated with a serial dilution of 10μM dBET1 or MZ1 PROTAC compounds for 1 hour. For both PROTACs, a dose-dependent increase in BRET ratio was observed. Error bars represent standard deviation, n = 3.

nanobret assay for protacs

Antibody-based assay for PROTAC activity. HEK293 cells containing endogenously tagged HiBiT-BET family members and expressing LgBiT were treated at the indicated times with 1 μM dBET1 or MZ1 PROTACs. Cells were lysed with digitonin and incubated for 10 min with both primary polyclonal anti-Ubiquitin and Alexa Fluor® 594 fluorescent secondary antibody to determine NanoBRET® ratios. Data are represented as fold increase in NanoBRET® values by normalizing to t = 0 time point. Variability expressed as SEM from n = 3 experiments.

Contact us to learn more about antibody approaches for studying target ubiquitination,
or get started with the NanoBRET® Ubiquitination Starter Kit.

Biochemical Approaches

We also offer biochemical methods to monitor ubiquitination of a target protein. The example shown here detects ubiquitination of the E3 ligase Cbl-b using Lumit® Immunoassay technology.

Lumit Cbl-b autoubiquitination assay

Schematic overview of a biochemical approach to monitor target protein ubiquitination. In the presence of necessary ubiquitination components E1, E2 and ATP, the Lumit® Immunoassay can measure the interaction between biotinylated ubiquitin and GST-Cbl-b.

measuring autoubiquitination of cbl-b dependence on ATP

Measuring autoubiquitination of Cbl-b. Panel A. A concentration-dependent increase in Cbl-b-GST ubiquitination in the presence of ATP causes increase in luminescence signal. As the process is ATP-dependent, in the absence of ATP there is a low, background signal. Panel B. A dilution series of unlabeled ubiquitin was used to compete off the biotinylated ubiquitin and results in concentration-dependent decrease in luminescence.

To learn more about antibody-based approaches to study target protein ubiquitination, download our application note or contact us.

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