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Applications of a Bioluminescent Peptide Tag: Simple, Quantitative Protein Detection Down to Endogenous Levels

This Webinar introduces the HiBiT protein tagging system. Topics include:

  • Simple detection workflows using lytic, surface-expression and blotting methods
  • Options for expressing HiBiT-tagged proteins, including knocking into endogenous loci with a clone-free CRISPR editing protocol
  • How HiBiT is used to study regulated protein abundance, targeted protein degradation, receptor internalization and protein trafficking

 

Summary

Regulated protein expression, degradation and trafficking to the cell surface are fundamental elements of normal cellular physiology, and protein dysregulation underlies many disease states.  Monitoring changes in protein abundance usually involves SDS-PAGE followed by immunoblotting, a labor-intensive process that requires high-quality antibodies. We have used NanoLuc® Binary Technology (NanoBiT), a two-part complementation system based on NanoLuc® luciferase, to develop a novel peptide tag for sensitively quantifying bioluminescent proteins with no antibody requirement. The peptide tag, designated High BiT (HiBiT), is only 11 amino acids in length. HiBiT-tagged proteins are measured using detection reagents containing its complementing polypeptide Large BiT (LgBiT), which binds with high affinity to HiBiT (KD~1nM) reconstituting a bright, luminescent enzyme. The HiBiT tag uses a simple bioluminescent method to quantify total or surface expression of proteins while offering a wide dynamic range and sensitivity compatible with detecting endogenously expressed proteins when combined with CRISPR gene editing.


Speaker

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Christopher Eggers, PhD
Senior Research Scientist

Dr. Christopher Eggers received his Ph.D. in biochemistry and molecular biology from the University of California at San Francisco and then completed a postdoctoral fellowship at the Howard Hughes Medical Institute at UC San Diego. Dr. Eggers has been a Senior Research Scientist at Promega since 2011, where he has principally focused on developing bioluminescent assays to measure protein abundance and interactions based on the NanoLuc® and NanoBiT® technologies. This includes leading the technical development of the Nano-Glo® Dual-Luciferase® Reporter Assay and the Nano-Glo® HiBiT Detection Systems.

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