Department of Biochemistry and Molecular Biology, Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Korea.
Department of Transdisciplinary Medicine, Seoul National University Hospital, Seoul 03080, Korea.
STAR Protoc. 2023 Dec 15;4(4):102748. doi: 10.1016/j.xpro.2023.102748. Epub 2023 Nov 23.
Proteasomes are heterogeneous in forms and functions, but how the equilibrium among the 20S, 26S, and 30S proteasomes is achieved and altered is elusive. Here, we present a protocol for purifying and characterizing proteasome species. We describe steps for generating stable cell lines; affinity purifying the proteasome species; and characterizing them through native PAGE, activity assay, size-exclusion chromatography, and mass spectrometry. These standardized methods may contribute to biochemical studies of cellular proteasomes under both physiological and pathological conditions. For complete details on the use and execution of this protocol, please refer to Choi et al. (2023)..
蛋白酶体在形式和功能上具有多样性，但 20S、26S 和 30S 蛋白酶体之间的平衡是如何实现和改变的还不得而知。在这里，我们提供了一种纯化和鉴定蛋白酶体种类的方案。我们描述了生成稳定细胞系的步骤；亲和纯化蛋白酶体种类；并通过天然 PAGE、活性测定、分子筛层析和质谱对其进行表征。这些标准化方法可能有助于在生理和病理条件下对细胞蛋白酶体进行生化研究。有关此方案使用和执行的完整详细信息，请参阅 Choi 等人。(2023)。
