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Target Degradation
Quantitative measurement of target protein levels fuels degrader discovery and optimization while deepening our understanding of cell biology and disease. HiBiT technology delivers ultrasensitive detection of protein degradation for high-throughput screens, and HiBiT knock-in cell lines work seamlessly with both endpoint and live-cell kinetic assays—providing the robust metrics needed to rank and refine the best degrader candidates.
For native cell models, Lumit® ImmunoAssay Cellular Systems use a labeled-antibody approach to quantify target-protein levels, allowing degrader efficacy measurements across a broad range of relevant cell backgrounds.
Is My Target Degraded?
Assessing Degradation Using HiBiT Knock-In Cell Lines
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 form a binary luminescent protein.Upon HiBiT–LgBiT engagement, the active luciferase protein produces a bright, highly sensitive readout that correlates with target protein levels. When HiBiT is introduced at the endogenous locus using CRISPR gene editing, it enables direct quantification of endogenous protein degradation. The HiBiT peptide tag itself can be detected with Anti-HiBiT mAb, expanding the detection options for HiBiT-tagged proteins.
Addition of compounds that elicit degradation results in loss of luminescence signal, which is highly quantitative and can be measured using endpoint or real-time methods to derive quantitative metrics of degrader compound properties.
An illustration of HiBiT fusion:LgBiT complementation inside a cell.
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. Panel A. Kinetic luminescence; Panel B. Degradation rate; C: Dmax.
Study protein degradation in real time using CRISPR knock-in cell lines and clones.
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HiBiT Knock-In Degradation Workflow
Both endpoint and live-cell analysis start with a cell line expressing the HiBiT-POI fusion, typically generated through CRISPR/Cas9 knock-in of the HiBiT tag to study the target protein under endogenous expression conditions.
Endpoint analysis of HiBiT-tagged protein levels provides a quantitative, high-throughput compatible method for determining degrader compound efficacy. Generation of cellular dose–response curves can be used to calculate DC50 and Dmax values and assess both on-target efficacy and off-target degradation.
Live-cell kinetic analysis of HiBiT-tagged protein levels is performed with intracellular expression of LgBiT. ViaScript LgBiT mRNA Delivery System allows for rapid uniform expression of intracellular LgBiT across a variety of cell lines. Luminescence is detected in real-time using Nano-Glo® Extended Live Cell Substrates and may be monitored on the GloMax® Discover instrument. The resulting data can be analyzed to derive quantitative compound parameters, such as degradation rate and duration at Dmax, using the Pronect™ TPD Kinetic App.
A greater understanding of the cellular response to degrader treatment can be obtained by visualizing with the GloMax® Galaxy Bioluminescence Imager.
Is My Target Degraded?
Assessing Degradation in Native (Non-Modified) Cells
We offer cellular methods to monitor degradation of a target protein in native cells. In this example, the Lumit® Immunoassay Cellular System is used to detect SMARCA 4 degradation.
Monitoring Targeted Protein Degradation with Lumit® Cellular Immunoassay
Degradation of native SMARCA4 following PROTAC treatment. 50,000 cells were seeded per well. After treatment with the indicated compounds, the Lumit® Immunoassay Cellular System (Set 2) was used to detect the level of SMARCA4. Panel A. SMARCA4 level after treatment with 250nM of ACBI-1, 250nM ACBI-1and 20µM MG132, 250nM Cis-ACBI-1, or DMSO for 5 hours. Panel B. Profiling SMARCA4 degradation with ACBI-1 in three different cell lines. Panel C. Time course of SMARCA4 degradation with ACBI-1.
Learn more about the Lumit® Immunoassay Cellular System.
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