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

Mitochondrial polymerase gamma (Pol?A) is the only replicative polymerase in mitochondria. To determine Pol?A ubiquitylation in cells, Flag-Pol?A and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-Pol?A is checked for ubiquitylation. Alternately, in vitro synthesized Pol?A and MITOL are used to determine whether Pol?A 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 Pol?A 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).
Mesh Terms:
DNA Polymerase gamma, Mitochondria, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination
STAR Protoc
Date: Dec. 16, 2022
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