Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells.
Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells.
复制标题
用于检测哺乳动物细胞中内源性和过度表达的过氧化物酶体蛋白的免疫标记。
DOI：
10.1007/978-1-0716-3048-8_4
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Schrader TA
中科院分区：
文献类型：
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作者：
Schrader TA
过氧化物酶体是哺乳动物体内一种动态的亚细胞器，在细胞脂质代谢和氧化还原平衡中起重要作用。它们在人类健康和疾病中发挥着广泛的功能，新的作用，机制和调控途径仍在发现中。最近阐明的过氧化物酶体的生物学作用包括作为抗病毒防御枢纽、细胞内信号平台、免疫调节剂和感觉细胞中的保护性细胞器。此外，过氧化物酶体是复杂的细胞器间相互作用网络的一部分，其涉及通过膜接触的代谢合作和串扰。通过免疫标记检测细胞内的内源性和/或过表达的蛋白质告知我们已知和推定的过氧化物酶体蛋白质的细胞器甚至亚细胞器定位。反过来，这可以用来表征实验操作对细胞中过氧化物酶体的形态、分布和/或数量的影响，这些是控制过氧化物酶体功能的关键特性。在这里，我们提出了一个协议，成功地在我们的实验室培养的哺乳动物细胞中的过氧化物酶体蛋白的免疫标记。我们目前的免疫荧光和转染技术以及试剂，以确定本地化的内源性和过表达的过氧化物酶体蛋白。
Peroxisomes are dynamic subcellular organelles in mammals, playing essential roles in cellular lipid metabolism and redox homeostasis. They perform a wide spectrum of functions in human health and disease, with new roles, mechanisms, and regulatory pathways still being discovered. Recently elucidated biological roles of peroxisomes include as antiviral defense hubs, intracellular signaling platforms, immunomodulators, and protective organelles in sensory cells. Furthermore, peroxisomes are part of a complex inter-organelle interaction network, which involves metabolic cooperation and cross talk via membrane contacts. The detection of endogenous and/or overexpressed proteins within a cell by immunolabelling informs us about the organellar and even sub-organellar localization of both known and putative peroxisomal proteins. In turn, this can be exploited to characterize the effects of experimental manipulations on the morphology, distribution, and/or number of peroxisomes in a cell, which are key properties controlling peroxisome function. Here, we present a protocol used successfully in our laboratory for the immunolabelling of peroxisomal proteins in cultured mammalian cells. We present immunofluorescence and transfection techniques as well as reagents to determine the localization of endogenous and overexpressed peroxisomal proteins.