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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.


Second dimensional SDS-PAGE of complex I subunits together with those of complex V and complex III. The blue native gel strip was placed on top of the second dimensional SDS gel (10% resolving). Gel was stained by CBB staining.
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Figure 2: Second dimensional SDS-PAGE of complex I subunits together with those of complex V and complex III. The blue native gel strip was placed on top of the second dimensional SDS gel (10% resolving). Gel was stained by CBB staining.

Mentions: For the resolution of each individual complex I subunit, we initially took two approaches. The first one was to turn the whole gel strip 90 degrees counter clockwise and layered it onto the second dimensional gel. As shown in Figure 2, this approach separated not only complex I subunits, but also those of complexes V and III, among others. The second approach was to excise the complex I band from the blue native gel, and placed this band onto a second dimensional gel, resulting in the resolution of only complex I subunits as shown in Figure 3 whereby both CBB staining and silver staining of individual complex I proteins were demonstrated. This approach was preferred because it would facilitate band matching and excision between stained gels and Western blot membranes as no other complexes were present.


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)

Second dimensional SDS-PAGE of complex I subunits together with those of complex V and complex III. The blue native gel strip was placed on top of the second dimensional SDS gel (10% resolving). Gel was stained by CBB staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Second dimensional SDS-PAGE of complex I subunits together with those of complex V and complex III. The blue native gel strip was placed on top of the second dimensional SDS gel (10% resolving). Gel was stained by CBB staining.
Mentions: For the resolution of each individual complex I subunit, we initially took two approaches. The first one was to turn the whole gel strip 90 degrees counter clockwise and layered it onto the second dimensional gel. As shown in Figure 2, this approach separated not only complex I subunits, but also those of complexes V and III, among others. The second approach was to excise the complex I band from the blue native gel, and placed this band onto a second dimensional gel, resulting in the resolution of only complex I subunits as shown in Figure 3 whereby both CBB staining and silver staining of individual complex I proteins were demonstrated. This approach was preferred because it would facilitate band matching and excision between stained gels and Western blot membranes as no other complexes were present.

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.