Department of Obstetrics and Gynecology, C.S. Mott Center for Human Growth and Development, Wayne State University, Detroit, MI, USA.
Methods Mol Biol. 2021;2255:1-12. doi: 10.1007/978-1-0716-1162-3_1.
Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.
细胞凋亡是一种由级联生化事件诱导的程序性细胞死亡，其特征为细胞收缩、细胞膜起泡、染色质浓缩和 DNA 片段化等明显的形态变化。细胞凋亡由一类称为半胱氨酸蛋白酶的胱氨酸蛋白酶执行。半胱氨酸蛋白酶最初合成时为无活性的原酶，通过一系列切割反应被激活。活性半胱氨酸蛋白酶切割细胞底物，因此是细胞凋亡途径的主要效应因子。通过 Western blot 分析检测半胱氨酸蛋白酶的切割是证明诱导细胞凋亡的一种常规方法。在细胞凋亡的背景下，要正确分析 Western blot 结果，需要理解半胱氨酸蛋白酶在激活过程中的加工机制和结果。在本章中，我们描述了 Western blot 分析细胞凋亡过程中半胱氨酸蛋白酶切割的分步方法。我们详细介绍了蛋白提取、定量、凝胶电泳和 Western blot 分析 caspase-8 和 caspase-9 激活的方案。所述方法可应用于任何特定感兴趣的蛋白质。
