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

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 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.......

Materials

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