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

Wu J, Luo X, Yan LJ - Front Physiol (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center Fort Worth, TX, USA.

ABSTRACT
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 non-gradient 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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.

No MeSH data available.


Non-gradient blue native gel electrophoresis of cardiac mitochondria isolated from rat heart. (A) Gel image after electrophoresis without further CBB staining; (B) complex I activity staining by NADH and NBT. An 8% resolving gel was performed.
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Figure 1: Non-gradient blue native gel electrophoresis of cardiac mitochondria isolated from rat heart. (A) Gel image after electrophoresis without further CBB staining; (B) complex I activity staining by NADH and NBT. An 8% resolving gel was performed.

Mentions: Cardiac mitochondria were used in our condition set-up experiments (Figures 1–3). For complex I isolation, we used a non-gradient blue native PAGE method as previously described (Yan and Forster, 2009). Figure 1A shows complex I band localization after gel electrophoresis. It was not necessary to further stain the gel after electrophoresis as complex I was always visible due to its pre-binding of Coomassie blue achieved during sample preparation. Figure 1B shows in-gel complex I activity staining by incubating the gels strips in a solution containing NADH and NBT.


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

Wu J, Luo X, Yan LJ - Front Physiol (2015)

Non-gradient blue native gel electrophoresis of cardiac mitochondria isolated from rat heart. (A) Gel image after electrophoresis without further CBB staining; (B) complex I activity staining by NADH and NBT. An 8% resolving gel was performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374453&req=5

Figure 1: Non-gradient blue native gel electrophoresis of cardiac mitochondria isolated from rat heart. (A) Gel image after electrophoresis without further CBB staining; (B) complex I activity staining by NADH and NBT. An 8% resolving gel was performed.
Mentions: Cardiac mitochondria were used in our condition set-up experiments (Figures 1–3). For complex I isolation, we used a non-gradient blue native PAGE method as previously described (Yan and Forster, 2009). Figure 1A shows complex I band localization after gel electrophoresis. It was not necessary to further stain the gel after electrophoresis as complex I was always visible due to its pre-binding of Coomassie blue achieved during sample preparation. Figure 1B shows in-gel complex I activity staining by incubating the gels strips in a solution containing NADH and NBT.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center Fort Worth, TX, USA.

ABSTRACT
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 non-gradient 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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.

No MeSH data available.