Protocol to differentiate glycosylphosphatidylinositol-anchored prion protein from pro-prion protein in cancer cells

Huan Li; Jie Yang; Jingfeng Li; Zhenxing Gao; Jiang Xu; Chaoyang Li

doi:10.1016/j.xpro.2023.102298

Protocol to differentiate glycosylphosphatidylinositol-anchored prion protein from pro-prion protein in cancer cells

STAR Protoc. 2023 Jun 12;4(2):102298. doi: 10.1016/j.xpro.2023.102298. Online ahead of print.

Authors

Huan Li¹, Jie Yang², Jingfeng Li², Zhenxing Gao², Jiang Xu³, Chaoyang Li⁴

Affiliations

¹ Affiliated Cancer Hospital and Institute of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong High Education Institute, 78 Heng Zhi Gang Road, Guangzhou 510095, China. Electronic address: lihuanlyj@163.com.
² Affiliated Cancer Hospital and Institute of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong High Education Institute, 78 Heng Zhi Gang Road, Guangzhou 510095, China.
³ Department of Stomatology, First Affiliated Hospital, School of Medicine, Shihezi University, No. 107 North 2nd Road, Shihezi, Xinjiang 832008, China.
⁴ Affiliated Cancer Hospital and Institute of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong High Education Institute, 78 Heng Zhi Gang Road, Guangzhou 510095, China. Electronic address: chaoyangli@gzhmu.edu.cn.

Abstract

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).¹.

Keywords: Antibody; Cancer; Cell Biology; Flow Cytometry/Mass Cytometry; Protein Biochemistry.