Methods for Quantitative Assessment of Protein Degradation

Assessment of small molecules that promote selective protein degradation (degraders) requires detailed characterization and measurement of protein levels in cells. Here we describe ratio-metric methods based on a dual fluorescent GFP/mCherry reporter system to quantify cellular protein levels. We further develop a kinetic framework for the analysis of such data. We describe two methods of generating the stable GFP-protein of interest (POI)/mCherry reporter cell lines, alternative readout methods by FACS and Laser Scanning Cytometry as well as the corresponding tools used for processing and analysis of such data. Finally, we show that the commonly used half-maximal degradation constant (DC₅₀) or maximum degradation efficacy (D_max) metrics are time-dependent and propose a time-invariant Michaelis-Menten-like analysis of degradation kinetics with analogous key parameters K_{m app} and V_{max app}.