Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines

Ubiquitin is covalently conjugated to phospholipids as well as proteins; however, ubiquitinated phospholipids are less abundant than free ubiquitin and ubiquitinated proteins. Here, we describe protocols to purify ubiquitinated phospholipids in budding yeast and human cells based on their hydrophobi...

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Autores principales: Sakamaki, Jun-ichi, Mizushima, Noboru
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9758488/
https://www.ncbi.nlm.nih.gov/pubmed/36520633
http://dx.doi.org/10.1016/j.xpro.2022.101935
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author Sakamaki, Jun-ichi
Mizushima, Noboru
author_facet Sakamaki, Jun-ichi
Mizushima, Noboru
author_sort Sakamaki, Jun-ichi
collection PubMed
description Ubiquitin is covalently conjugated to phospholipids as well as proteins; however, ubiquitinated phospholipids are less abundant than free ubiquitin and ubiquitinated proteins. Here, we describe protocols to purify ubiquitinated phospholipids in budding yeast and human cells based on their hydrophobicity. Ubiquitinated phospholipids are purified by Triton X-114 phase partitioning and affinity purification and verified by phospholipase D treatment. These protocols enable the detection of tagged as well as endogenous mono- and poly-ubiquitinated phospholipids by immunoblotting. For complete details on the use and execution of this protocol, please refer to Sakamaki et al..(1)
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spelling pubmed-97584882022-12-18 Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines Sakamaki, Jun-ichi Mizushima, Noboru STAR Protoc Protocol Ubiquitin is covalently conjugated to phospholipids as well as proteins; however, ubiquitinated phospholipids are less abundant than free ubiquitin and ubiquitinated proteins. Here, we describe protocols to purify ubiquitinated phospholipids in budding yeast and human cells based on their hydrophobicity. Ubiquitinated phospholipids are purified by Triton X-114 phase partitioning and affinity purification and verified by phospholipase D treatment. These protocols enable the detection of tagged as well as endogenous mono- and poly-ubiquitinated phospholipids by immunoblotting. For complete details on the use and execution of this protocol, please refer to Sakamaki et al..(1) Elsevier 2022-12-13 /pmc/articles/PMC9758488/ /pubmed/36520633 http://dx.doi.org/10.1016/j.xpro.2022.101935 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Sakamaki, Jun-ichi
Mizushima, Noboru
Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
title Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
title_full Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
title_fullStr Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
title_full_unstemmed Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
title_short Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
title_sort protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9758488/
https://www.ncbi.nlm.nih.gov/pubmed/36520633
http://dx.doi.org/10.1016/j.xpro.2022.101935
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