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.
Protocol
1. Preparation of Reagent 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 …
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Materials
Dulbecco's Modified Eagle Medium
Life Technologies
11995-092
Fetal Bovine Serum
Life Technologies
10082147
Lipofectamine 2000
Life Technologies
11668019
MG132
Sigma-Aldrich
M8699
Amino acids
Sigma-Aldrich
Amino acids are used for making low fluorecence culturing medium
Cycloheximide
Sigma-Aldrich
C7698
Olympus IX-81 Inverted Fluorescence Microscope
Olympus
IX71/IX81
96 Well Black TC Plate w/ Transluscent Clear Bottom
Sigma-Greiner
89135-048
Fluorescence Bottom Plate Reader Infinite 200® PRO
Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins. J. Vis. Exp. (Pending Publication), e21224, doi: (2023).