Buntenbroich Ira, Simões Tânia, Escobar-Henriques Mafalda
Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.
Bio Protoc. 2021 Nov 20;11(22):e4225. doi: 10.21769/BioProtoc.4225.
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 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.
在本实验方案中，我们描述了使用合成阻断法对蛋白质随时间的稳定性进行分析。例如，我们在酵母中表达HA标签的酵母线粒体融合蛋白Fzo1，并通过环己酰亚胺（CHX）抑制翻译。作为一个可选步骤，在添加CHX之前，先用MG132进行蛋白酶体抑制。通过三氯乙酸（TCA）沉淀提取蛋白质，随后通过SDS-PAGE进行分离。使用HA特异性抗体进行免疫印迹和抗体标记以检测Fzo1。我们采用了用环己酰亚胺阻断蛋白质翻译的方法来分析高分子量蛋白质的稳定性，包括翻译后修饰及其对蛋白质周转的影响。
