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Targeted Protein Degradation

The brightness and small size of NanoLuc® luciferase and HiBiT peptide make them ideal protein fusion tags that provide sensitive and quantitative methods for monitoring changes in intracellular protein abundance even at endogeneous levels.

Investigating Key Stages in PROTAC™-Mediated Cellular Protein Degradation

Proteolysis targeting chimeras or PROTACs induce selective protein degradation by chemically linking a protein target with an the E3 ubiquitin ligase component, initiating degradation of the protein target via the ubiquitin-proteosome system (UPS). Interrogating the key steps required for effective PROTAC™-mediated target protein degradation remains a challenge, particularly in cells.

NanoLuc® technologies provide a platform of live-cell assays that can be used to measure real-time degradation kinetics and the key cellular interactions along the UPS that mediate the degradation process. You can even study endogenous protein response when HiBiT is inserted using CRISPR gene editing.

Overview of Live-Cell Based Protein Degradation by PROTACs
This illustration highlights questions about the protein degradation steps in live cells that we can answer with our bioluminescent and energy transfer technologies.

Answering Key Questions to Characterize PROTACs and Their Cellular Protein Interactions

  1. Is my target protein degraded?
    This protocol uses target proteins fused to NanoLuc® luciferase or the HiBiT protein tag to visualize protein degradation.
  2. Does my PROTAC enter the cell and bind to the target protein or E3 component?
    This protocol uses the NanoBRET™ Target Engagement (TE) Assay to evaluate PROTAC permeability and measure VHL binding.
  3. Does my PROTAC form a ternary complex with its target and the E3 recruiter?
    This protocol describes using the NanoBRET™ PPI Assay, an energy transfer method, to measure binding among a PROTAC, an E3 ligase and a target protein.
  4. Is my target protein ubiquitinated?
    This protocol uses the proximity-based NanoBRET™ PPI Assay to determine if ubiquitin is added to the target protein.
  5. Is my target protein trafficked to the proteasome?
    This protocol describes using the NanoBRET™ PPI Assay to measure the interaction between the target protein and proteasome.

Understanding and Optimizing PROTACs to Study Protein Degradation in Cells

Developing PROTACs that effectively target proteins for degradation can be challenging. Examining only the end result, protein degradation, provides little information about where along the ubiquitin proteasomal pathway the compound failed.

This peer-reviewed article shows how to characterize PROTACs in live cells using endogenous protein tagging and real-time assays that provide better metrics for degradation and examine key cellular protein interactions involved along the pathway. These tools can be used to guide more effective PROTAC™ development.

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NanoLuc Live Cell Assays for PROTAC Protein Degradation

How can I start characterizing PROTACs and protein degradation? Learn More