Reder Alexander, Michalik Stephan, Gerth Ulf
Interfaculty Institute for Genetics and Functional Genomics, Greifswald University, Greifswald, Germany.
Department of Microbial Physiology and Molecular Biology, Institute for Microbiology, University Greifwald, Greifswald, Germany.
Methods Mol Biol. 2018;1841:131-140. doi: 10.1007/978-1-4939-8695-8_10.
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.
特定蛋白质的半衰期变化很大，从几分钟到几小时，再到数天、数周、数年，甚至是生物体的整个生命周期（例如哺乳动物眼睛中的α-晶状体蛋白）。因此，通过蛋白水解来控制蛋白质活性是任何生物体最显著且明确的决定，因为恢复被破坏的蛋白质活性需要进行“昂贵的”重新合成。为了区分蛋白质降解与合成及积累，唯一可考虑的方法是脉冲追踪标记。在我们看来，追踪单个蛋白质命运的最准确方法是放射性脉冲追踪标记结合免疫沉淀法。除了详细描述标准方案外，本文还将讨论该方法的普遍适用性以及某些改进之处。
