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

This video describes an in vitro fluorescence-based microplate assay to study the degradation kinetics of a fluorescently-labeled misfolded luciferase mutant protein expressed in the nuclei of transfected mammalian cells. The assay involves the treatment of cells expressing the fluorescent mutant protein with a translation inhibitor and a proteasome inhibitor to assess the proteasome-mediated degradation of the misfolded protein.

1. ​Preparation of Reagent

1. Prepare low-fluorescence DMEM medium for assays using a microplate fluorescence reader. Mix 25 mM glucose, 0.4 mM glycine, 0.4 mM arginine, 0.2 mM cysteine, 4.0 mM glutamine, 0.2 mM histidine, 0.8 mM isoleucine, 0.8 mM leucine, 0.8 mM lysine, 0.2 mM methionine, 0.4 mM phenylalanine, 0.4 mM serine, 0.8 mM threonine, 0.078 mM tryptophan, 0.4 mM tyrosine, 0.8 mM valine, 1.8 mM CaCl₂, 0.81 mM MgSO₄, 5.33 mM KCl, 44.0 mM NaHCO₃, 110 mM NaCl, 0.9 mM .......

Figure 1: Fluorescent microplate-based assay for NLS-LucDM-GFP degradation. HeLa cells seeded on 96 well plates were transfected with 0.05 µg (A) or 0.1 µg (B) NLS-LucDM-GFP and treated with CHX in the presence or absence of MG132. (A and B) Fluorescence intensities of wells were measured at the indicated time points (M.......