Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL
Summary: 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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722005901 |
| _version_ | 1811292507847262208 |
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| author | Mansoor Hussain Shabnam Saifi Aftab Mohammed Sagar Sengupta |
| author_facet | Mansoor Hussain Shabnam Saifi Aftab Mohammed Sagar Sengupta |
| author_sort | Mansoor Hussain |
| collection | DOAJ |
| description | Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-13T04:46:29Z |
| format | Article |
| id | doaj.art-e418c10871a844d093073a174355ec39 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-13T04:46:29Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-e418c10871a844d093073a174355ec392022-12-22T03:01:50ZengElsevierSTAR Protocols2666-16672022-12-0134101710Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOLMansoor Hussain0Shabnam Saifi1Aftab Mohammed2Sagar Sengupta3National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, IndiaNational Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, IndiaNational Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, IndiaNational Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India; Corresponding authorSummary: 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). : 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/S2666166722005901Cell biologyGeneticsMolecular biologyProtein biochemistryProtein expression and purification |
| spellingShingle | Mansoor Hussain Shabnam Saifi Aftab Mohammed Sagar Sengupta Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL STAR Protocols Cell biology Genetics Molecular biology Protein biochemistry Protein expression and purification |
| 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 | Cell biology Genetics Molecular biology Protein biochemistry Protein expression and purification |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722005901 |
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