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Selectively targeting proteins for removal from the cell, instead of inhibiting protein activity, is a newer modality for potential therapy. The protein is targeted for degradation using the cell’s natural ubiquitin proteasome pathway (UPS). Compounds such as molecular glues and proteolysis targeting chimeras (PROTACs) initiate this process by linking the target protein to an E3 ligase. The cell’s UPS does the rest.

Promega offers a comprehensive selection of CRISPR-edited cell line pools and clones to facilitate studying popular protein degradation targets, using sensitive bioluminescence technology.

View Available Cell LinesDownload List (PDF)

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Key Questions To Consider When Developing Protein Degraders

Promega offers a portfolio of cell-based assays for use in development of effective protein degraders. These products are used to answer the following key questions:


Key questions when developing protein degraders
key stages in protac mediation

Is My Target Degraded?

HiBiT technology enables quantitative analysis of protein degrader function. HiBiT is an 11-amino acid peptide tag which has high affinity for its complementary partner, LgBiT. Together, they comprise the binary luminescent protein, NanoBiT® Luciferase. Upon HiBiT-LgBiT engagement, the active luciferase protein produces a very bright and highly sensitive readout correlated to the endogenous target protein level, when HiBiT is introduced at the endogenous locus using CRISPR gene editing.

Addition of compounds that elicit degradation results in loss of luminescence signal, which is highly quantitative and can be measured in real time. Cellular dose-response curves can be obtained and monitored over a 24- to 48-hour time frame, allowing for accurate determination of degradation rate, Dmax, DC50 values and protein recovery. 

This approach allows rapid rank ordering of degradation against a series of different parameters, and the assay is suitable for high-throughput screening.

HiBit:LgBiT complementation

An illustration of HiBiT fusion:LgBiT complementation inside a cell.

Degradogram with MZ1
Rate calculation with MZ1
DC50 calculation with MZ1

Degradation kinetics of endogenous HiBiT-BRD4 following PROTAC treatment.  HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line. Cells were treated with a titration of MZ1 in CO2-independent medium containing Nano-Glo® Endurazine™ substrate. A: Kinetic luminescence; B: Degradation rate; C: Dmax.

Study protein degradation in real time using CRISPR knock-in cell lines and clones.
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What Is the PROTAC's Cellular Permeability and Target Affinity?

When developing degrader compounds, it is important to assess their cellular permeability and affinity for target proteins and E3 ligases. The NanoBRET™ Target Engagement (TE) Assays enable the measurement of protein-small molecule binding interactions in live cells, providing quantitative determination of compound affinity and occupancy for target proteins. 

The NanoBRET™ TE assays utilize bioluminescent resonance energy transfer (BRET) from a NanoLuc® luciferase-tagged target protein and a cell-permeable fluorescent NanoBRET™tracer. When the tracer reversibly engages the target-NanoLuc® fusion protein in cells, BRET is achieved by luminescent energy transfer from NanoLuc®-tagged target protein to the cell-permeable fluorescent tracer. Target engagement is measured by competition of compound with the NanoBRET™ tracer, which results in a loss of BRET.  The measurement produces a cellular affinity for the compound and can be performed using an addition only, scalable, multi-well plate method.

We have successfully developed assays using the NanoBRET™ TE technology for multiple target classes important in drug discovery, including kinases, histone deacetylases (HDAC) and bromodomains. Recently, the technology has been applied to E3 ligases and NanoBRET™ TE assays have been developed for CRBN, VHL, XIAP, cIAP and MDM2. The NanoBRET™ TE E3 ligase assays can be performed in live or permeabilized cells, which allows the user to assess compound affinity for the E3 ligase as well as compound permeability. Learn more about NanoBRET™ TE E3 ligase assays in this poster.

Residence time is an important parameter to monitor during SAR studies to improve degradation potency. NanoBRET™ TE Assays can be configured in a kinetic mode to understand residence time of a compound for its target protein.

Tools are also available to build NanoBRET™ TE Assays for additional E3 ligases and other targets. See Designing Your Own Target Engagement Assay.

target engagement assay principle


Principle of the NanoBRET™ Target Engagement Assay

BRD4 NanoBRET TE live cells
CRBN NanoBRET TE live and permeabilized cells

Measuring cellular affinity of BET degraders targeting BRD4 using the NanoBRET™ TE Assay. Comparison of cellular binding affinity for two related PROTACs that target BRD4 (left panel) using a BRD4 NanoBRET™ TE assay. Degrader affinity for the E3 ligase CRBN was compared in live and permeabilized cells using a NanoBRET™ TE CRBN assay to assess contribution of compound permeability to measured binding affinity (right panel). The study revealed that dBET6 is more permeable than dBET1 and dBET6 has slightly higher affinity for CRBN compared to dBET1.

Currently available E3 ligase assays include: CRBN and VHL

Through our Elite Access program, we also offer assays for XIAP, cIAP and MDM2.

Does My PROTAC Form a Ternary Complex?

Formation of the E3 ternary complex (containing the target, degradation compound and E3 ligase) is a crucial step in targeted protein degradation. This step represents a critical parameter in the development and optimization of effective degrader compounds. NanoBRET™ technology is ideally suited to study ternary complex formation in a live-cell format, allowing for both endpoint or kinetic analysis.

In this assay, target protein serves as the energy donor (bioluminescence), expressed in the cell as an exogenous transient NanoLuc® fusion or an endogenously tagged HiBiT fusion in a LgBiT-expressing cell. HaloTag® fusions with von Hippel-Lindau (VHL) or cereblon (CRBN) E3 ligase components are expressed exogenously and labeled with fluorescent ligand to serve as the energy acceptor.

PROTAC Ternary Complex Formation

A schematic depiction of NanoBRET™ ternary complex formation.

brd4-vhl interaction
brd4-vhl interactions
BRD4/VHL and ABRD4/CRBN ternary complex formation

Multiplexing PROTAC-induced BRD4 VHL/CRBN ternary complex formation and BRD4 protein levels using live-cell endpoint detection. Cells (BRD4/VHL assay) transfected with a 1:100 ratio of NanoLuc®-BRD4 donor plasmid to HaloTag®-VHL or HaloTag®-CRBN acceptor plasmid, pretreated with MG132 (or DMSO control), subsequently treated with 1µM MZ1 (VHL-based PROTAC) or DMSO. The NanoBRET™ ratio indicates ternary complex formation (Panel A) and NanoLuc® luminescence indicates target protein levels (Panel B) caused by PROTAC treatment.

Kinetically monitoring BRD4/VHL ternary complex formation following PROTAC treatment. 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®-VHL acceptor plasmid. Cells were pretreated with MG132 prior to PROTAC treatment. NanoBRET™ signal was measured using the NanoBRET™ Nano-Glo® Kinetic Detection System to monitor ternary complex formation in real time.

Get started with ternary complex studies for VHL and CRBN.
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Does My Target Become Ubiquitinated?

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-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.

What Is the Phenotypic Consequence of Target Degradation? 

Temporal degradation of proteins inside a cell often elicits a much different phenotype than genetic knockouts or protein mutations. HaloPROTAC3, a fusion of a HaloTag® label and a PROTAC, is a rapid and highly effective way to understand and characterize protein degradation phenotype.

HaloPROTAC3 recruits an endogenous VHL E3 ligase component to a HaloTag® fusion protein, resulting in ubiquitination and degradation via the proteasome pathway. HaloPROTAC contains a degradation-inducing acylamine moiety, coupled to a chloroalkane moiety by a linker of variable length.

To study endogenous protein loss in relevant cell backgrounds, we recommend incorporating a HaloTag® or HiBiT-HaloTag® tag into the target protein loci via CRISPR/Cas9 gene editing. Loss of protein is monitored in cells treated with HaloPROTAC-3 either using HaloTag® monoclonal antibodies (for HaloTag® tags) or live-cell luminescence (with HiBiT-HaloTag® tags). HaloPROTAC3 shows fast burst loss that is sustained over time with endogenously tagged HaloTag® fusion proteins.

HaloPROTAC overview

Schematic overview of HaloPROTAC function.

BRD4 degradogram

The HiBiT-BRD4 degradogram. BRD4, endogenously tagged with HiBiT and HaloTag® label, is degraded using increasing concentrations of HaloPROTAC and quantified using  HiBiT luminescence.

HaloPROTAC3 is compared to dTag technologies in the 2020 Cell paper Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery ( The paper describes the field of proteolysis-targeting chimeras (PROTACs), which are capable of modulating protein concentrations at a post-translational level using the ubiquitin-proteasome system. HaloPROTAC3 is also compared to PROTAC-mediated protein level modulation with other technologies such as RNAi and genome editing.

HaloPROTAC3 has also been utilized in vivo (mice) to degrade PNPLA3 levels in liver. For additional details refer to the publication: Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis

Ready to start your degradation assays with HaloPROTAC3?
 View HaloPROTAC Ligands

What Degradation Profiling Services Does Promega Offer?

Endpoint degradation compound profiling
  • Single concentration or DRC to determine DC50
  • Cell viability recommended at late time point

HEK293 CRISPR HiBiT CDK family members were treated with pan-kinase PROTAC TL-12-186 and relative levels of degradation were determined by monitoring fractional loss of luminescence at the designated time point (left panel). Cell viability was additionally monitored with Cell-Titer® Glo and plotted relative to degradation (right panel).

CDK panel
CDK panel

Kinetic degradation compound profiling

  • Recommended time frame is 0-24 hours
  • Single concentrations or DRC to determine Dmax50
  • Provides rate of degradation, Dmax, Dmax50 and recovery information

CRISPR IKZF1-HiBiT Jurkat cells were treated with a DRC of Iberdomide and live-cell, kinetic degradation was monitored on the GloMax® Discover. Kinetic degradation of IKZF1 was additionally performed with several other molecular glues and from those profiles, degradation rates (left panel) and Dmax (right panel) were determined across the DRC. The comparative response to each compound is plotted for degradation rate and Dmax.

kinetic degradation panel
kinetic degradation panel

NanoBRET™ Target Engagement

  • Selectivity profiling against >200 kinase proteins and compound dose-response follow-ups
  • E3 Ligase compound profiling (CRBN, VHL, XIAP, cIAP1, MDM2)

NanoBRET™ Target Engagement competitive displacement experiments in HEK293 live cells expressing NanoLuc® fusions of either CRBN or VHL E3 ligase components. CRBN assays show displacement by molecular glue compounds and CRBN-based PROTACs (left panel), while VHL assays show displacement by a VHL binder and VHL-based PROTACs (right panel).

NanoBRET target engagement assay
nanobret target engagement

NanoBRET™ ternary complex formation

  • Determine complex association rate and potency

CRISPR HiBiT-BRD2 HEK293 cells expressing LgBiT were transfected with HaloTag-CRBN and treated with the indicated concentrations of dBET1 (left panel) or dBET6 (right panel) and MG132. Kinetic live-cell NanoBRET™ experiments were performed, reflecting the induced BRD2:dBET:CRBN ternary complex formation over the indicated time frame.


NanoBRET™ target ubiquitination

  • Determine ubiquitination rate and potency

CRISPR HiBiT-BRD2 HEK293 cells expressing LgBiT were transfected with HaloTag-Ub and treated with the indicated concentrations of dBET1 (left panel) or dBET6 (right panel). Kinetic live-cell NanoBRET™ experiments were performed, reflecting the induced ubiquitination of BRD2 over the indicated time frame.


Get started with our targeted protein degradation profiling services.
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