Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment

Abstract

Summary: N-Glycosylation is a common protein modification catalyzed by a series of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. Here, based on a previously established Golgi α-mannosidase-I-deficient cell line, we present a protocol to investigate the enzymatic activity of exogenously expressed Golgi α-mannosidase IA in interphase and mitotic cells. We describe steps for cell surface lectin staining and subsequent live cell imaging. We also detail PNGase F and Endo H cleavage assays to analyze protein glycosylation.For complete details on the use and execution of this protocol, please refer to Huang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell-based Assays
• Microscopy