Protein degradation

Tools Accelerating Targeted Protein Degradation Research

Our partner, LifeSensors, is a global leader in ubiquitin biology and targeted protein degradation. Beyond their widely adopted TUBEs that we previously introduced here : Ubiquitination & Targeted Protein Degradation Tools, LifeSensors offers efficient tools enabling the discovery, validation, and mechanistic characterization of PROTACs, molecular glues, and DUBTACs, accelerating the discovery of novel molecules facilitating targeted protein degradation. 

 

PROTACs (Proteolysis-targeting Chimeras)

PROTAC are hetero-bifunctional small molecules that contain two functional ligands connected via a linker; one ligand binds to a target protein and the other ligand binds to an E3 ligase (Figure 1).

Figure 1: PROTAC structure. The target protein of interest (POI) and the E3 ligase are linked via a linker. This leads to the POI ubiquitination.

PROTACs artificially hijack the components of the UPS (Ubiquitin Proteasome System) to degrade a target protein. By bringing the target protein and the E3 ligase into proximity, the POI is ubiquitinated and degraded via the proteasome (Figure 2).

The use of PROTACs for drug discovery approaches became a major therapeutic strategy for previously “undruggable” proteins in oncology, immunology, and neurodegeneration - opening a new therapeutic avenue across multiple disease areas by allowing the elimination of proteins that cannot be inhibited by classical small molecules and a rapid and reversible control of protein levels.

Figure 2: PROTACs mechanism of action. PROTACs bind the E3 ligase and the protein of interest (T) leading to its ubiquitination and degradation via the proteasome.

However, this mechanism remains complex and the PROTAC discovery platform faces several challenges with an unmet need for functional assays that can study the effects of PROTACs on the ubiquitination of targets and their degradation. LifeSensors has developed high-throughput methods to directly monitor PROTAC mediated ubiquitylation and degradation of target proteins, including functional ubiquitination and degradation assays in cells. PROTAC-mediated ubiquitination in vitro permits rapid screening of compound libraries and allows simplification of the medicinal chemistry approach to rationally design potent molecules.

Molecular Glue

Molecular glues are small molecules that stabilize interactions between a target protein and an E3 ligase (Figure 3). 

Figure 3: Molecular glue structure.

They have the same function as PROTACs and are divided in two classes:

  1. Glues that bind to an E3 ligase to change its conformation so that it binds and ubiquitinates a neo-substrate
  2. Glues that bind to a target protein, change its conformation such that the structure is altered, exposing lysines to be ubiquitinated by neighboring ligases. Depending upon the type of ligase employed for ubiquitination, if lysine 48 chains are built on the target protein, the proteasome will recognize the target and degrade it. If lysine 63 chains are built on the target protein, the protein may be trafficked to another compartment with a loss of function. However, lysine 63 chains most likely promote autophagy, and the protein will be degraded by lysosomal degradation mechanisms.

LifeSensors has a variety of tools to discover and tease out the mechanism of molecular glue mediated ubiquitination and degradation.

DUBTACs (Deubiquitinating targeting chimeras) 

DUBs are a large group of proteases that remove ubiquitin from proteins, rescuing ubiquitinated proteins from proteasome attack (Figure 4). They antagonize the E3 ligase activity, regulate protein trafficking and are implicated in epigenetic chromatin remodeling.

Figure 4: DUBs mechanism of action. 

DUBTACs consists of three components: (1) a DUB recruiter, (2) a target protein binder and (3) a linker connecting both entities. DUBTACs recruit DUBs to a target protein and remove ubiquitin chains, resulting in stabilization of target proteins, restoring protein levels, function, and their rescue from degradation via the proteasome.

Figure 5: DUBTACs mechanism of action

TUBEs: Tandem Ubiquitin Binding Entities

TUBEs are high-affinity “ubiquitin traps” engineered with multiple ubiquitin-binding domains arranged in tandem (Figure 2), allowing them to:

  • Bind polyubiquitin chains with sub-nanomolar affinity
  • Protect ubiquitinated proteins from endogenous DUBs, preserving true ubiquitination states
  • Selectively enrich specific chain types (K48, K63, M1/linear, pan-ubiquitin)
  • Capture ubiquitinated substrates without modifying ubiquitin or substrates

Figure 2: TUBEs structure. Tandem-domain architecture enabling high-affinity capture of polyubiquitin chains.

This tandem-domain architecture acts as a high-affinity molecular trap, enabling researchers to isolate native ubiquitin signals without introducing artifacts from tags, overexpression, or engineered reporter systems. 
They are ideal for:

  • Poly-ubiquitinated protein detection
  • Ubiquitinated protein purification and visualization
  • Targeted protein degradation studies
  • Investigating protein stabilization, signaling, and ubiquitination pathways

Why TUBEs Outperform Classical Anti-ubiquitin Antibodies?

Ubiquitin chains are not uniform. Their lysine linkage type and branching architecture encode distinct biological outcomes -degradation, signaling, trafficking, autophagy, or stress responses. LifeSensors’ TUBEs allows accurate mapping of K48 vs. K63 signaling (Figure 3).

Figure 3: Lysine-Ubiquitin linkage and different biological outcomes.

These TUBEs have as well superior sensitivity through nanomolar binding affinity as well as high specificity for polyubiquitin, drastically reducing false positives. Finally, they are fully compatible with western blotting, chemiluminescence, ELISA, fluorescence, proteomics, and mass spectrometry.

Supporting Reagents for Ubiquitin Research

LifeSensors provides a complete toolkit to complement TUBEs and enable deeper insights into protein regulation:

  • E1, E2, E3 Ligases: Allow understanding the mechanisms behind protein degradation and signaling, including downstream effects and potential off-target interactions 
  • Deubiquitinases (DUBs): A broad library of active DUB enzymes to support research into ubiquitination dynamics and enzyme activity 
  • Ubiquitin Chains: A variety of chain types, substrates for DUBs, including phosphorylated and biotinylated forms 

All of these products are biologically active proteins, ensuring robust results that synthetic alternatives cannot match. 

Ready to use TUBE based-ELISA tools (K48 & K63)

For screening and drug discovery, TUBEs are available in fully validated 96-well formats and in High-Throughput Screening (HTS)-compatible plate formats for large-scale screening, as well as other flexible formats such as magnetic TUBEs, Biotinylated TUBEs, Fluorescein- and TAMRA-TUBEs.

LifeSensors’ K63 Ubiquitin Linkage ELISA kit (#PA630) is intended to enrich and quantitate K63 ubiquitination in cellular and tissue lysates or in in vitro ubiquitination reactions. This kit enables relative and absolute quantitation of K63 ubiquitination on target proteins in cellular and tissue lysates under various treatments and conditions. The signal generated in this "sandwich" ELISA assay is a quantitative measure of K63-linked ubiquitination of target proteins.

Figure 4: K63 Ubiquitin Linkage ELISA Kit (Chain Selective) principle assay.

Case study– Kinase Inhibitor Validation Using PA630

 

Figure 5: Validation of kinase inhibitor that inhibits K63 polyubiquitination on RIPK2 kinase using LifeSensors K63 Ubiquitin linkage ELISA Kit.

 

L18-MDP treatment resulted in enhanced levels of K63 ubiquitination on RIPK2 compared to vehicle control sterile water when probed with anti-RIPK2 on PA630 K63 TUBE plates. RIPK2 K63-polyubiquitination mediated by L18-MDP can be reversed in presence of kinase inhibitors. Linkage specific K63-TUBE ELISA kit clearly demonstrated effect of kinase inhibitors on RIPK2 inhibition reaching 100% inhibition at 100-300 nM of kinase inhibitors with IC50 values ~5nM consistent with literature. The data clearly suggests that PA630 can be used to selectively monitor levels of K63 polyubiquitination in presence of a stimulation like L18-MDP followed by monitoring inhibitory effects to predict IC50s crucial for reliable drug discovery.

These kits are widely used as validation assays in PROTAC and molecular glue experiments. Please look out for new special pages focusing on these technologies based on TUBEs very soon! 

Why Choose LifeSensors?

  • Study ubiquitination and degradation of proteins simultaneously
  • Complete suite of validation studies to establish binding, ternary complex formation, and cellular degradation
  • High throughput-homogenous ubiquitination assays to screen for molecular glues
  • ~30 E3 ligases amenable to PROTAC discovery
  • Proteomics to characterize protein degradation and to establish selectivity.

Not a Biocide. Not a Disinfectant. Just Effective.

  • Prevents microbial growth through redox-active catalysis, no chemicals, no silver release.

  • Proven >99.9% germ reduction and biofilm prevention in independent laboratory testing.

  • Ideal for: water tanks, lab baths, humidifiers, incubators, cooling loops, RV systems, and more.

  • Free from biocides, nano-silver, pyrogens, toxins, and compliant with TVO, EPA, WHO limits.

Choose Your Pack Size

1 pouch — Prothermal PLUS (2601-PLUS)
Perfect for small systems, benchtop baths, incubator trays, and compact reservoirs.

10 pouches — Prothermal PLUS (2601-PLUS-10)
Ideal for multi-unit labs, shared facilities, and long-term rotational maintenance.