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

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 detail 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.