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

Abstract

Source: Guo, L. et. al., Assays for the Degradation of Misfolded Proteins in Cells. J. Vis. Exp. (2016)

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

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 NaH₂PO₄. Adjust the pH of the solution using HCl or NaOH to pH 7.4. Sterilize the medium through filtration.
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Results

Figure 1: Fluorescent microplate-based assay for NLS-LucDM-GFP degradation. HeLa cells .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	TECAN	Infinite 200® PRO
Prism 5	GraphPad		Statistical analysis software