Fractionation of Native Protein Complexes from Mammalian Cells to Determine the Differential Proteasome Activity and Abundance.
Fractionation of Native Protein Complexes from Mammalian Cells to Determine the Differential Proteasome Activity and Abundance.
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DOI：
10.21769/bioprotoc.4822
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发表时间：
2023-09-20
期刊：
影响因子：
0.8
通讯作者：
中科院分区：
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真核细胞具有不同类型、大小不同的蛋白酶体。最小的蛋白水解活性颗粒是20S蛋白酶体，它可以降解受损和氧化的蛋白质；最常见的较大颗粒是 26S 蛋白酶体，它可以降解泛素化蛋白质。 26S蛋白酶体由20S颗粒形成，其上覆盖有一个或两个调节颗粒，称为19S。虽然蛋白酶体颗粒在细胞质、内质网和细胞核中发挥作用，但我们对它们在不同细胞区室中的丰度和活性的了解仍然有限。我们提供了一个三步方案，首先涉及基于去污剂的细胞质和核区室的分级分离，保持蛋白酶体复合物的完整性和活性。其次，该方案采用天然凝胶分离级分中的大型多蛋白复合物，并对活性和不同蛋白酶体颗粒进行基于荧光的凝胶内定量。最后，该方案涉及蛋白质凝胶内变性并转移到 PVDF 膜上。然后，蛋白质印迹检测并定量不同的蛋白酶体颗粒。因此，该协议允许对来自不同细胞区室的单个蛋白酶体颗粒的活性和丰度进行灵敏测量。它已针对小鼠胚胎干细胞诱导的运动神经元进行了优化，但也可应用于多种哺乳动物细胞系。 主要特点 • 活性核和细胞质蛋白酶体复合物的分级分离方案。 • 基于天然电泳和荧光的凝胶内活性测定，可实现丙烯酰胺凝胶基质内活性复合物的可视化和定量。 • 凝胶内蛋白质变性，然后将复合物转移到 PVDF 膜上，这样可以使用抗体分析复合物的丰度。
Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines. Key features • Protocol for fractionation of active nuclear and cytoplasmic proteasome complexes. • Native electrophoresis and fluorescence-based in-gel activity assay, which allows the visualization and quantification of active complexes within the acrylamide gel matrix. • In-gel protein denaturation followed by transfer of complexes to PVDF membrane, which allows the analysis of complexes’ abundance using antibodies.
影响因子：
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作者：
Tang Y;Guo H;Vermeulen AJ;Heuck AP
通讯作者：
Heuck AP