Analyzing the topology of N-linked glycans by PNGase F accessibility assay
Summary: 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 th...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723004252 |
| _version_ | 1797769045949284352 |
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| author | Jingcheng Wang Jin Ye |
| author_facet | Jingcheng Wang Jin Ye |
| author_sort | Jingcheng Wang |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-12T21:02:08Z |
| format | Article |
| id | doaj.art-90847b6df3164dbfab62fbaa4ce232c6 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-12T21:02:08Z |
| publishDate | 2023-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-90847b6df3164dbfab62fbaa4ce232c62023-07-31T04:09:22ZengElsevierSTAR Protocols2666-16672023-09-0143102458Analyzing the topology of N-linked glycans by PNGase F accessibility assayJingcheng Wang0Jin Ye1Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Corresponding authorDepartment of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723004252Cell BiologyCell-based AssaysCell Separation/FractionationMolecular BiologyProtein Biochemistry |
| spellingShingle | Jingcheng Wang Jin Ye Analyzing the topology of N-linked glycans by PNGase F accessibility assay STAR Protocols Cell Biology Cell-based Assays Cell Separation/Fractionation Molecular Biology Protein Biochemistry |
| title | Analyzing the topology of N-linked glycans by PNGase F accessibility assay |
| title_full | Analyzing the topology of N-linked glycans by PNGase F accessibility assay |
| title_fullStr | Analyzing the topology of N-linked glycans by PNGase F accessibility assay |
| title_full_unstemmed | Analyzing the topology of N-linked glycans by PNGase F accessibility assay |
| title_short | Analyzing the topology of N-linked glycans by PNGase F accessibility assay |
| title_sort | analyzing the topology of n linked glycans by pngase f accessibility assay |
| topic | Cell Biology Cell-based Assays Cell Separation/Fractionation Molecular Biology Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723004252 |
| work_keys_str_mv | AT jingchengwang analyzingthetopologyofnlinkedglycansbypngasefaccessibilityassay AT jinye analyzingthetopologyofnlinkedglycansbypngasefaccessibilityassay |