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Updated: Jun 24, 2025
Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
Protocol for the comprehensive biochemical and cellular profiling of small-molecule degraders using CoraFluor TR-FRET
N Connor Payne1, Saki Ichikawa2, Christina M Woo3
1Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA.
Abstract:
Comprehensive characterization of small-molecule degraders, including binary and ternary complex formation and degradation efficiency, is critical for bifunctional ligand development and understanding structure-activity relationships. Here, we present a protocol for the biochemical and cellular profiling of small-molecule degraders based on CoraFluor time-resolved fluorescence resonance energy transfer (TR-FRET) technology. We describe steps for labeling antibodies and proteins, tracer saturation binding, binary target engagement, ternary complex profiling, and off-rate determination. We then detail procedures for the quantification of endogenous and GFP fusion proteins in cell lysates. For complete details on the use and execution of this protocol, please refer to Ichikawa et al.1.
