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

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dc.creatorWu, Jinzi
dc.creatorLuo, Xiaoting
dc.creatorYan, Liang-Jun
dc.date.accessioned2019-08-22T19:35:49Z
dc.date.available2019-08-22T19:35:49Z
dc.date.issued2015-03
dc.date.submitted2015-03-04T12:47:47-08:00
dc.description.abstractAbstract 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. Keywords: blue native/SDS-PAGE, diabetes, 4-hydroxynonenal, mitochondria, reactive oxygen species, streptozotocin
dc.identifier.urihttps://hdl.handle.net/20.500.12503/26360
dc.language.isoen
dc.titleTwo dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE)
dc.typeposter
dc.type.materialtext

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