Abstract
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
Original language | English (US) |
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Article number | 102458 |
Journal | STAR Protocols |
Volume | 4 |
Issue number | 3 |
DOIs | |
State | Published - Sep 15 2023 |
Keywords
- Cell Biology
- Cell Separation/Fractionation
- Cell-based Assays
- Molecular Biology
- Protein Biochemistry
ASJC Scopus subject areas
- General Neuroscience
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology