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.

In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the tagged protein using anti-HA affinity beads. Subsequently, we separate the higher molecular weight (ubiquitylated) forms of Fzo1 via SDS-PAGE. Finally, immunoblotting and immunodecoration are used to detect the protein and its ubiquitylated forms using an HA-specific antibody. By using this protocol, it is possible to separate and visualize higher molecular weight forms of low abundant proteins such as Fzo1 and detect sharp and distinct bands above the unmodified protein by Western blot.