Kaushal Varsha, Herzog Christian, Haun Randy S, Kaushal Gur P
Biology Department, Hendrix College, Conway, AR, USA.
Methods Mol Biol. 2014;1133:141-54. doi: 10.1007/978-1-4939-0357-3_9.
Members of the caspase family of proteases are evolutionarily conserved cysteine proteases that play a crucial role as the central executioners of the apoptotic pathway. Since the discovery of caspases, many methods have been developed to detect their activation and are widely used in basic and clinical studies. In a mouse tissue, caspase activation can be monitored by cleavage of caspase-specific synthetic substrates and by detecting cleaved caspase by western blot analysis of the tissue extract. In tissue sections, active caspase can be detected by immunostaining using specific antibodies to the active caspase. In addition, among the myriads of caspase-specific substrates known so far, cleaved fragments produced by caspases from the substrates such as PARP, lamin A, and cytokeratin-18 can be monitored in tissue sections by immunostaining as well as western blots of tissue extracts. In general, more than one method should be used to ascertain detection of activation of caspases in a mouse tissue.
半胱天冬酶蛋白酶家族成员是进化上保守的半胱氨酸蛋白酶，作为细胞凋亡途径的核心执行者发挥着关键作用。自从发现半胱天冬酶以来，已经开发出许多方法来检测它们的激活，并且广泛应用于基础和临床研究。在小鼠组织中，半胱天冬酶的激活可以通过半胱天冬酶特异性合成底物的切割以及通过对组织提取物进行蛋白质印迹分析来检测切割后的半胱天冬酶进行监测。在组织切片中，可以使用针对活性半胱天冬酶的特异性抗体通过免疫染色来检测活性半胱天冬酶。此外，在目前已知的众多半胱天冬酶特异性底物中，半胱天冬酶从诸如聚（ADP-核糖）聚合酶、核纤层蛋白A和细胞角蛋白-18等底物产生的切割片段，可以通过组织切片的免疫染色以及组织提取物的蛋白质印迹进行监测。一般来说，应该使用不止一种方法来确定小鼠组织中半胱天冬酶激活的检测。
