Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL

Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and...

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Autores principales: Hussain, Mansoor, Saifi, Shabnam, Mohammed, Aftab, Sengupta, Sagar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9508469/
https://www.ncbi.nlm.nih.gov/pubmed/36136752
http://dx.doi.org/10.1016/j.xpro.2022.101710
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author Hussain, Mansoor
Saifi, Shabnam
Mohammed, Aftab
Sengupta, Sagar
author_facet Hussain, Mansoor
Saifi, Shabnam
Mohammed, Aftab
Sengupta, Sagar
author_sort Hussain, Mansoor
collection PubMed
description Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and MITOL are used to determine whether PolγA is ubiquitylated. Either anti-ubiquitin or anti-Flag antibody is used to detect the ubiquitylated product. Thus, we provide a detailed, reliable, highly reproducible protocol for detecting ubiquitylation of PolγA by MITOL, both in cells and in vitro. For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021).
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spelling pubmed-95084692022-09-25 Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL Hussain, Mansoor Saifi, Shabnam Mohammed, Aftab Sengupta, Sagar STAR Protoc Protocol Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and MITOL are used to determine whether PolγA is ubiquitylated. Either anti-ubiquitin or anti-Flag antibody is used to detect the ubiquitylated product. Thus, we provide a detailed, reliable, highly reproducible protocol for detecting ubiquitylation of PolγA by MITOL, both in cells and in vitro. For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021). Elsevier 2022-09-21 /pmc/articles/PMC9508469/ /pubmed/36136752 http://dx.doi.org/10.1016/j.xpro.2022.101710 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Hussain, Mansoor
Saifi, Shabnam
Mohammed, Aftab
Sengupta, Sagar
Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
title Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
title_full Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
title_fullStr Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
title_full_unstemmed Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
title_short Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
title_sort protocol to detect in vitro and in cell ubiquitylation of mitochondrial dna polymerase gamma by mitochondrial e3 ligase mitol
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9508469/
https://www.ncbi.nlm.nih.gov/pubmed/36136752
http://dx.doi.org/10.1016/j.xpro.2022.101710
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