Protocol to differentiate glycosylphosphatidylinositol-anchored prion protein from pro-prion protein in cancer cells.
Protocol to differentiate glycosylphosphatidylinositol-anchored prion protein from pro-prion protein in cancer cells.
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DOI：
10.1016/j.xpro.2023.102298
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发表时间：
2023-06-12
期刊：
影响因子：
--
通讯作者：
Li, Chaoyang
中科院分区：
文献类型：
--
作者：
Li, Huan;Yang, Jie;Li, Jingfeng;Gao, Zhenxing;Xu, Jiang;Li, Chaoyang
糖基磷脂酰肌醇（GPI）-锚合成的缺陷导致功能改变的前蛋白质的产生。然而，缺乏用于功能分析的前蛋白特异性抗体。在这里，我们提出了一个协议，以区分GPI锚定的朊蛋白（PrP）从前PrP在癌细胞中使用的互补方法适用于其他GPI锚定蛋白。我们首先描述了磷脂酰肌醇特异性磷脂酶C治疗和基于流式细胞术的检测步骤。然后，我们详细介绍了羧肽酶Y（CPDY）测定，包括抗体固定，亲和纯化，CPDY处理，和基于蛋白质印迹的检测。有关本方案使用和执行的完整详细信息，请参见Li等人。（2022年）。区分癌细胞中GPI锚定的PrP和pro-PrP的补充方法进行PI-PLC处理和流式细胞术检测的步骤CPDY处理分析和蛋白质印迹检测的详细信息适用于区分其他GPI锚定的蛋白质及其前体蛋白出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。糖基磷脂酰肌醇（GPI）-锚合成的缺陷导致功能改变的前蛋白质的产生。然而，缺乏用于功能分析的前蛋白特异性抗体。在这里，我们提出了一个协议，以区分GPI锚定的朊蛋白（PrP）从前PrP在癌细胞中使用的互补方法适用于其他GPI锚定蛋白。我们首先描述了磷脂酰肌醇特异性磷脂酶C治疗和基于流式细胞术的检测步骤。然后，我们详细介绍了羧肽酶Y（CPDY）测定，包括抗体固定，亲和纯化，CPDY处理，和基于蛋白质印迹的检测。
Defects of glycosylphosphatidylinositol (GPI)-anchor synthesis lead to the production of pro-proteins with altered functions. However, pro-protein-specific antibodies for functional analysis are lacking. Here, we present a protocol to differentiate GPI-anchored prion protein (PrP) from pro-PrP in cancer cells using a complementary approach applicable to other GPI-anchored proteins. We first describe steps for phosphatidylinositol-specific phospholipase C treatment and flow-cytometry-based detection. We then detail the carboxypeptidase Y (CPDY) assay including antibody immobilization, affinity purification, CPDY treatment, and western-blot-based detection. For complete details on the use and execution of this protocol, please refer to Li et al. (2022). A complementary approach to differentiate GPI-anchored PrP from pro-PrP in cancer cells Steps to perform PI-PLC treatment and detection by flow cytometry Details on CPDY treatment assay and detection by western blot Applicable to differentiate other GPI-anchored proteins from their pro-proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Defects of glycosylphosphatidylinositol (GPI)-anchor synthesis lead to the production of pro-proteins with altered functions. However, pro-protein-specific antibodies for functional analysis are lacking. Here, we present a protocol to differentiate GPI-anchored prion protein (PrP) from pro-PrP in cancer cells using a complementary approach applicable to other GPI-anchored proteins. We first describe steps for phosphatidylinositol-specific phospholipase C treatment and flow-cytometry-based detection. We then detail the carboxypeptidase Y (CPDY) assay including antibody immobilization, affinity purification, CPDY treatment, and western-blot-based detection.
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