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Analysis of Protein Stability by Synthesis Shutoff
Ira Buntenbroich1, Tânia Simões1, Mafalda Escobar-Henriques1
1Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.
Bio-Protocol
|December 15, 2021
Summary
This protocol analyzes protein stability using synthesis shutoff. Cycloheximide (CHX) blocks translation to assess protein turnover, including high molecular weight proteins and post-translational modifications.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Protein stability is crucial for cellular function.
- Understanding protein turnover aids in studying disease mechanisms.
- Existing methods may not fully capture the dynamics of high molecular weight proteins.
Purpose of the Study:
- To describe a protocol for analyzing protein stability over time using synthesis shutoff.
- To demonstrate the application of this method for studying yeast mitofusin Fzo1.
- To adapt cycloheximide-mediated translation inhibition for high molecular weight protein analysis.
Main Methods:
- Protein synthesis was inhibited using cycloheximide (CHX).
- Optional proteasomal inhibition with MG132 was performed.
- Proteins were extracted using trichloroacetic acid (TCA) precipitation.
- SDS-PAGE, immunoblotting, and antibody detection were used to analyze HA-tagged Fzo1.
Main Results:
- The protocol successfully assessed the stability of HA-tagged yeast mitofusin Fzo1.
- The method allows for the analysis of protein turnover in response to translation inhibition.
- Adaptation enables the study of high molecular weight proteins and their modifications.
Conclusions:
- Synthesis shutoff with cycloheximide is an effective method for protein stability analysis.
- This protocol is valuable for investigating protein turnover, including complex proteins.
- The method provides insights into post-translational modifications and their impact on protein stability.
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