Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor

Proper cellular proteostasis is essential to cellular fitness and viability. Exogenous stress conditions compromise proteostasis and cause aggregation of cellular proteins. We have developed a fluorogenic sensor (AgHalo) to quantify stress-induced proteostasis deficiency. The AgHalo sensor uses a destabilized HaloTag variant to represent aggregation-prone cellular proteins and is equipped with a series of fluorogenic probes that exhibit a fluorescence increase when the sensor forms either soluble oligomers or insoluble aggregates. Herein, we present protocols that describe how the AgHalo sensor can be employed to visualize and quantify proteome stress in live cells using a direct fluorescence read-out and visualization with a fluorescence microplate reader and a microscope. Additionally, protocols for using the AgHalo sensor in combination with fluorogenic probes and commercially available HaloTag probes to enable two-color imaging experiments are described. These protocols will enable use of the AgHalo sensor to visualize and quantify proteostasis in live cells, a task that is difficult to accomplish using previous, always-fluorescent methods. © 2018 by John Wiley & Sons, Inc.