Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins
Published: April 30, 2023
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 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…
Divulgazioni
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 | TECAN | Infinite 200® PRO | |
| Prism 5 | GraphPad | Statistical analysis software |
Tags
Citazione di questo articolo
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).