Protocol for the quantitative identification of endogenously ISGylated proteins from mammalian cell lines.
| Autor: | Wardlaw CP; Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address: wardlawc@mskcc.org., Miele MM; Proteomics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA., Li Z; Proteomics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA., Hendrickson RC; Proteomics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA., Petrini JHJ; Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address: petrinij@mskcc.org. |
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| Jazyk: | angličtina |
| Zdroj: | STAR protocols [STAR Protoc] 2024 Mar 15; Vol. 5 (1), pp. 102843. Date of Electronic Publication: 2024 Jan 29. |
| DOI: | 10.1016/j.xpro.2024.102843 |
| Abstrakt: | Ubiquitin-like protein ISG15 plays an important role in an array of cellular functions via its covalent attachment to target proteins (ISGylation). Here, we present a protocol for the identification of ISGylated proteins that avoids the caveats associated with ISG15 overexpression and minimizes the likelihood of false positives. We describe steps for the tagging of endogenous ISG15, followed by genotyping and clone selection. We then detail steps for ISGylation induction, the isolation of ISGylated proteins, and their identification via quantitative mass spectrometry. For complete details on the use and execution of this protocol, please refer to Wardlaw and Petrini. 1 . Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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