Analysis of Protein Stability by Synthesis Shutoff

Ira Buntenbroich; Tânia Simões; Mafalda Escobar-Henriques

doi:10.21769/BioProtoc.4225

Analysis of Protein Stability by Synthesis Shutoff

Bio Protoc. 2021 Nov 20;11(22):e4225. doi: 10.21769/BioProtoc.4225.

Authors

Ira Buntenbroich¹, Tânia Simões¹, Mafalda Escobar-Henriques¹

Affiliation

¹ Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.

Abstract

In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover.

Keywords: Cycloheximide chase; Fzo1; SDS-PAGE; Shutoff; Western blot.