ハラ　ツカサ   Hara Tsukasa   原　司    所属   医学研究科 医学研究科 (医学部医学科をご参照ください)    職種   特任助教
論文種別	原著
言語種別	英語
査読の有無	査読あり
表題	Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV.
掲載誌名	正式名：Biology open 略　 称：Biol Open ISSNコード：20466390/20466390
掲載区分	国外
巻・号・頁	9(1),pp.bio047936
著者・共著者	Hara Tsukasa, Shibata Yuma, Amagai Ryosuke, Okado-Matsumoto Ayako
担当区分	筆頭著者
発行年月	2020/01
概要	The in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.
DOI	10.1242/bio.047936
PMID	31852667