拟南芥原生质体瞬时表达系统中蛋白质稳定性研究方案

Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System.

作者信息

Planchais Séverine, Camborde Laurent, Jupin Isabelle

机构信息

Laboratoire de Virologie Moléculaire, Institut Jacques Monod, CNRS, UMR 7592, Univ. Paris-Diderot, Sorbonne Paris Cité, Paris, France.

Adaptation des Plantes aux Contraintes Environnementales, UMR 7618, IEES Paris, Univ. Pierre-et-Marie-Curie, Paris, France.

出版信息

Methods Mol Biol. 2016;1450:175-94. doi: 10.1007/978-1-4939-3759-2_14.

DOI:10.1007/978-1-4939-3759-2_14

PMID:27424754

Abstract

Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in details two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions.

摘要

蛋白质稳定性影响生物学的许多方面，在体内测量其稳定性可为生物系统提供重要见解。本章详细描述了两种基于特定蛋白质在拟南芥原生质体中的瞬时表达来评估其稳定性的方法。首先，一种基于细胞蛋白质放射性代谢标记的脉冲追踪分析，随后对感兴趣的蛋白质进行免疫沉淀。随着时间的推移监测放射性信号的下降，并可用于确定蛋白质的半衰期。另外，我们还介绍了一种基于使用报告蛋白的非放射性分析方法，其比例可以定量。该分析可用于确定特定条件下感兴趣蛋白质的相对稳定性。