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Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation
Overview
This video describes a degradation assay for misfolded proteins using cycloheximide treatment along with detergent fractionation. This method aids in studying the dynamics of misfolded proteins and uncovering the in-depth mechanisms of protein turnover.
Protocol
1. Preparation of Reagent
- Prepare cell lysis buffer (50 mM Tris, pH 8.8, 100 mM NaCl, 5 mM MgCl2, 0.5% NP-40). Supplement 2 mM DTT, 1x complete protease cocktail, and 250 IU/ml benzonase before use.
- Prepare pellet buffer (20 mM Tris, pH 8.0, 15 mM MgCl2). Supplement 2 mM DTT, 1x complete protease cocktail, and 250 IU/ml benzonase before use.
- Prepare 3x boiling buffer (6% SDS, 20 mM Tris, pH 8.0). Supplement 150 mM DTT before use.
- Prepare low-fluorescence DME.......
Representative Results
Figure 1. Detection of Atxn1 82Q-GFP by fluorescent microscopy and detergent fractionation. (A) HeLa cells were transfected with Atxn1 82Q-GFP and stained with DAPI. Individual and merged images are shown. Scale bar = 10 µm. (B) A diagram showing the detergent fractionation of Atxn1-82Q expressing cells as described in protocol 2. .......
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Source: Guo, L. et al., Assays for the Degradation of Misfolded Proteins in Cells. J. Vis. Exp. (2016).
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