Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins

Nuclear misfolded proteins lacking a defined tertiary structure are tagged with small ubiquitin-like modifiers, SUMOs, and ubiquitin molecules, which facilitate their degradation by the proteolytic system, including the ubiquitin-proteasome system, the UPS.

In several neurodegenerative diseases, UPS dysfunction causes the accumulation and aggregation of nuclear misfolded proteins, leading to neuronal death.

To investigate the degradation rate of nuclear misfolded proteins in vitro, treat a culture of adherent mammalian cells with a mixture comprising a fluorescent-labeled misfolded protein-encoding plasmid DNA and a transfection reagent.

Successfully transfected cells express fluorescent-labeled, short half-life misfolded proteins tagged with a nuclear localization signal. Upon transportation to the nucleus, these misfolded proteins undergo SUMOylation and ubiquitination and are transferred to the UPS for protein degradation and clearance.

Post-incubation, replace the medium with a low-fluorescence medium containing cycloheximide. Cycloheximide, a cell-permeable molecule, binds to the large ribosomal unit and blocks the elongation phase of protein translation and further protein synthesis, facilitating the study of the degradation rate of the fluorescent-labeled translated misfolded proteins. 

Treat a set of wells with a proteasome inhibitor to block the proteasome activity and cause the misfolded proteins to accumulate. Using a microplate reader, measure the fluorescence of the misfolded proteins.

A time-dependent decline in the fluorescence indicates misfolded protein degradation. Cells treated with the proteasome inhibitor exhibit similar fluorescence intensity over time, denoting the inhibition of misfolded protein degradation.

After seeding and transfecting HeLa cells in 96-well plates according to the text protocol; 20 to 24 hours after transfection, examine the cells for GFP expression. Remove the medium at approximately 200 microliters of 1x PBS to each well, and then, aspirate it to remove the residual DMEM.

Add 60 microliters of low-fluorescence DMEM with 5% FBS, and 50 micrograms per milliliter of cycloheximide, and add 10 micromolar MG132 to one set of samples.

With a fluorescence plate reader, measure the GFP signal reading the plates every hour for up to 8 to 10 hours. Export the data, and carry out statistical analysis according to the text protocol.