National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
STAR Protoc. 2022 Dec 16;3(4):101710. doi: 10.1016/j.xpro.2022.101710. Epub 2022 Sep 21.
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).
线粒体聚合酶 γ（PolγA）是线粒体中唯一具有复制功能的聚合酶。为了确定细胞中 PolγA 的泛素化，过表达 Flag-PolγA 和 MITOL，然后检查免疫沉淀的 Flag-PolγA 是否发生泛素化。或者，使用体外合成的 PolγA 和 MITOL 来确定 PolγA 是否被泛素化。可以使用抗泛素或抗 Flag 抗体来检测泛素化产物。因此，我们提供了一种详细、可靠、高度可重复的方案，用于检测 MITOL 对 PolγA 的泛素化，既可以在细胞内，也可以在体外进行。有关该方案使用和执行的完整详细信息，请参阅 Hussain 等人（2021 年）。
