Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE)

Frontiers in Physiology

Xiaoting eLuo¹  Jinzi eWu²  Liang-Jun eYan² 

[1] Gannan Medical University;University of North Texas Health Science Center;

关键词: Mitochondria;    Reactive Oxygen Species;    diabetes;    Streptozotocin;    4-hydroxynonenal;    Blue native/SDS-PAGE;   

DOI  :  10.3389/fphys.2015.00098

来源: DOAJ

【 摘 要 】

The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional SDS-PAGE. HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

【 授权许可】

Unknown