Department of Obstetrics and Gynecology, C.S. Mott Center for Human Growth and Development, Wayne State University, Detroit, MI, USA.
Methods Mol Biol. 2021;2255:21-26. doi: 10.1007/978-1-0716-1162-3_3.
Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.
在细胞内，蛋白质根据其功能和激活状态被分隔到不同的细胞器中。在受到刺激后，翻译后修饰或细胞器膜完整性的丧失会导致蛋白质从一个隔室转移到另一个隔室。这种蛋白质的运动或蛋白质易位对蛋白质功能有显著影响。在细胞凋亡的情况下，这种情况表现得非常明显，线粒体驻留蛋白细胞色素 C 的细胞质易位启动了细胞凋亡途径的内在途径的激活。在实验中，可以通过亚细胞分级分离和随后对分离的级分进行 Western blot 分析来证明蛋白质易位。本章描述了从细胞沉淀中获得线粒体和细胞质级分并确定其纯度和完整性的逐步过程。
