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

Summary 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