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Monitoring Functional Mechanisms of Protein Degradation in Living Cells

Part # PS330

Abstract

Kristin Riching, Sarah Mahan, James Vasta, Mark McDougall, Cesear Corona, Chad Zimprich, Matt Robers, Danette L. Daniels, and Marjeta Urh
Promega Corporation, 2800 Woods Hollow Rd, Madison WI 53711 USA

Here, we present a live-cell, luminescence-based technology platform that enables characterization of PROTAC compound mechanism of action using either ectopic or endogenous target expression formats. We employ CRISPR/Cas9 endogenous tagging of target proteins with the small peptide, HiBiT, which has high affinity for and can complement with the LgBiT protein to produce NanoBiT® luminescence. This allows for sensitive detection of endogenous protein levels in living cells, and can also serve as a BRET energy donor to study protein:protein or protein:small molecule interactions.

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