Analyzing the topology of N-linked glycans by PNGase F accessibility assay

While N-glycans are synthesized in the lumens, some of them reach the cytosolic side of membranes through retro-translocation independent of endoplasmic-reticulum-associated degradation. Here, we present a protocol to measure the topology of N-glycans in a transmembrane protein, based on the principle that cytosolic but not luminal N-glycans are trimmed by PNGase F in the absence of detergent. We describe the procedures for this protocol consisting of microsome preparation from cells, PNGase F accessibility assay, and western blot analysis. For complete details on the use and execution of this protocol, please refer to Wang et al.1 [Display omitted] •Protocol for identifying localization of N-glycans on intracellular organelle proteins•Steps for preparing microsomes and performing PNGase F accessibility assay•Assay to measure the deglycosylation of proteins in microsomes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. While N-glycans are synthesized in the lumens, some of them reach the cytosolic side of membranes through retro-translocation independent of endoplasmic-reticulum-associated degradation. Here, we present a protocol to measure the topology of N-glycans in a transmembrane protein, based on the principle that cytosolic but not luminal N-glycans are trimmed by PNGase F in the absence of detergent. We describe the procedures for this protocol consisting of microsome preparation from cells, PNGase F accessibility assay, and western blot analysis.