In Vivo Detection of Oxidized Proteins: A Practical Approach to Tissue-Derived Mitochondria - Protein Misfolding and Cellular Stress in Disease and Aging - page 249

7. Running buffer (10X): 250 mM Tris, 1.92 M glycine, 1%
(w/v) SDS.
8. Prestained molecular weight marker: 7-175 kDa (New
England Biolabs, Ipswich, MA, USA).

2.4. Western Blot
of DNP-Derivatized
Proteins

1. Methanol from Merck.
2. Phosphate Buffered Saline (PBS) 10× (Invitrogen).
3. Transfer buffer: 250 mM Tris, 1,92 M glycine, 20% Methanol
(v/v). Store at 4°C.
4. Hybond-ECL nitrocellulose membrane (GE Healthcare,
Uppsala, Sweden).
5. Filter papers (3MM, Whatman)
6. PBS with Tween (PBS-T): dilute 10× PBS to 1× in sterile
water with the addition of 0.5% Tween-20 (v/v).
7. Red Ponceau S (Sigma Aldrich) solution: dissolve at 0.1%
(w/v) in 1% (v/v) acetic acid.
8. Blocking buffer and antibody dilution buffer: 5% (w/v) non-
fat dry milk in PBS-T.
9. Rabbit polyclonal anti-DNPH antibody (Molecular Probe,
Invitrogen), mouse monoclonal antibody against respiratory
chain complex I 39 kDa subunit (NDUFA9) and anti-porin
antibody (Invitrogen).
10. Secondary antibodies: anti-mouse and anti-rabbit IgG, horse-
radish peroxidase (HRP)-linked species-specific whole anti-
bodies (GE Healthcare).
11. Enhanced Chemiluminescence (ECL) HRP Substrate Reagent
(GE Healthcare) and ECL hyperfilms (GE Healthcare).

3. Methods

Proteins are one of the major targets of oxygen-free radicals and
other reactive species and the oxidative modification can modu-
late biochemical characteristics of proteins such as enzymatic
activity, DNA-binding activities of transcription factors, and the
susceptibility to proteolytic degradation. Metal catalyzed oxida-
tion of proteins introduces carbonyl groups (aldehydes and
ketones) at lysine, arginine, proline or threonine residues in a site-
specific manner ( 13, 14 ). The experimental procedure described
in this chapter allows simple and sensitive immunodetection of
carbonyl groups, providing information on the oxidation status
of proteins.
The carbonyl groups in the protein side chains are derivatized
to 2,4 dinitrophenylhydrazone (DNP-hydrazone) by reaction