Mitochondrial Medicine

0554565 - FGÚ 2022 RIV US eng M - Monography Chapter
Kohutiar, M. - Eckhardt, Adam
A Method for Analysis of Nitrotyrosine-Containing Proteins by Immunoblotting Coupled with Mass Spectrometry.
Mitochondrial Medicine. Vol. 2. New York: Springer, 2021 - (Weissig, V.; Edeas, M.), s. 383-396. Methods in Molecular Biology, 2276. ISBN 978-1-0716-1266-8
R&D Projects: GA MZd(CZ) NV17-31564A
Institutional support: RVO:67985823
Keywords : mitochondria * nitrotyrosine * 2D electrophoresis * immunoblotting * mass spectrometry
OECD category: Biochemical research methods
https://link.springer.com/protocol/10.1007/978-1-0716-1266-8_28

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.
Permanent Link: http://hdl.handle.net/11104/0329274