The fluorescent protein stability assay: An efficient method for monitoring intracellular protein stability

Abstract

The stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs. {Graph presented} METHOD SUMMARY To analyze the stability of a specific protein, a lentiviral vector has been developed that allows expression of three different proteins from a single mRNA: FLAG-tagged enhanced green fluorescent protein, the puromycin resistance factor and FLAG-tagged red fluorescent protein fused to the protein of interest. Lentiviral particles obtained fromthe vector are used to establish polyclonal ormonoclonal cell lines by puromycin resistance selection. These cells efficiently express both green and hybrid red fluorescent proteins at similar levels, and measurement of the fluorescence ratio by flow cytometry allows a precise assessment of the stability of the protein of interest under various physiological and drug-induced conditions.