Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting
Summary: Ubiquitin-fold modifier 1 (UFM1) system is a recently identified ubiquitin-like modification with essential biological functions. Similar to ubiquitination, the covalent conjugation of UFM1 (UFMylation) to target proteins involves a three-step enzymatic cascade catalyzed sequentially by UFM...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-03-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166721007802 |
| _version_ | 1818318860656312320 |
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| author | Junzhi Zhou Qian Liang Maogong Dong Xiaohe Ma Yaqi Jin Di Guan Jiang Liu Miao Wang Yu-sheng Cong |
| author_facet | Junzhi Zhou Qian Liang Maogong Dong Xiaohe Ma Yaqi Jin Di Guan Jiang Liu Miao Wang Yu-sheng Cong |
| author_sort | Junzhi Zhou |
| collection | DOAJ |
| description | Summary: Ubiquitin-fold modifier 1 (UFM1) system is a recently identified ubiquitin-like modification with essential biological functions. Similar to ubiquitination, the covalent conjugation of UFM1 (UFMylation) to target proteins involves a three-step enzymatic cascade catalyzed sequentially by UFM1-activating enzyme 5 (UBA5, E1), UFM1-conjugating enzyme 1 (UFC1, E2), and UFM1-specific ligase 1 (UFL1, E3). Here, we provide an optimized protocol adapted to previously reported methods for detecting the UFMylation of target protein in human cells and in vitro assays, respectively, with high reliability and reproducibility.For complete details on the use and execution of this protocol, please refer to Liu et al. (2020). |
| first_indexed | 2024-12-13T09:59:56Z |
| format | Article |
| id | doaj.art-3519c53c76654c809ba9081cc7efdb78 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-13T09:59:56Z |
| publishDate | 2022-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-3519c53c76654c809ba9081cc7efdb782022-12-21T23:51:41ZengElsevierSTAR Protocols2666-16672022-03-0131101074Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblottingJunzhi Zhou0Qian Liang1Maogong Dong2Xiaohe Ma3Yaqi Jin4Di Guan5Jiang Liu6Miao Wang7Yu-sheng Cong8Key Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, ChinaKey Laboratory of Aging and Cancer Biology of Zhejiang Province, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, China; Corresponding authorSummary: Ubiquitin-fold modifier 1 (UFM1) system is a recently identified ubiquitin-like modification with essential biological functions. Similar to ubiquitination, the covalent conjugation of UFM1 (UFMylation) to target proteins involves a three-step enzymatic cascade catalyzed sequentially by UFM1-activating enzyme 5 (UBA5, E1), UFM1-conjugating enzyme 1 (UFC1, E2), and UFM1-specific ligase 1 (UFL1, E3). Here, we provide an optimized protocol adapted to previously reported methods for detecting the UFMylation of target protein in human cells and in vitro assays, respectively, with high reliability and reproducibility.For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).http://www.sciencedirect.com/science/article/pii/S2666166721007802Cell BiologyMolecular BiologyProtein Biochemistry |
| spellingShingle | Junzhi Zhou Qian Liang Maogong Dong Xiaohe Ma Yaqi Jin Di Guan Jiang Liu Miao Wang Yu-sheng Cong Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting STAR Protocols Cell Biology Molecular Biology Protein Biochemistry |
| title | Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting |
| title_full | Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting |
| title_fullStr | Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting |
| title_full_unstemmed | Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting |
| title_short | Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting |
| title_sort | optimized protocol to detect protein ufmylation in cells and in vitro via immunoblotting |
| topic | Cell Biology Molecular Biology Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166721007802 |
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