Tsukasa HARA    Department   Research Institutes and Facilities, Research Institutes and Facilities    Position   Assistant Professor (Fixed Term)
Article types	Original article
Language	English
Peer review	Peer reviewed
Title	Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV.
Journal	Formal name：Biology open Abbreviation：Biol Open ISSN code：20466390/20466390
Domestic / Foregin	Foregin
Volume, Issue, Page	9(1),pp.bio047936
Author and coauthor	Hara Tsukasa, Shibata Yuma, Amagai Ryosuke, Okado-Matsumoto Ayako
Authorship	Lead author
Publication date	2020/01
Summary	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