How to assess protein stability: Half-life determination of a regulatory protein in bacillus subtilis

Abstract

The half-life of a particular protein is highly variable, reaching from minutes to hours, over days and weeks to years or even a whole life time of an organism (e.g., α-crystalline of the mammalian eye). Thus, controlling protein activity by proteolysis is the most dramatic and unambiguous decision by any organism, because reconstitution of the destroyed protein activity requires an “expensive” new synthesis. To distinguish degradation from protein synthesis and accumulation only one method comes into consideration—pulse-chase labeling. In our hands, the most accurate method to track the fate of a single protein is radioactive pulse-chase labeling combined with immunoprecipitation. Besides a detailed description of the standard protocol, the general applicability as well as certain improvements of the method will be discussed here.