In Vivo Detection of Oxidized Proteins: A Practical Approach to Tissue-Derived Mitochondria - Protein Misfolding and Cellular Stress in Disease and Aging - page 254

Fig. 2. Increased level of oxidized mitochondrial proteins in Afg3l2 mutant mice. Carbonyl levels after reaction with DNPH
are measured by Western blot on mitochondrial-enriched preparations from brain from both Afg3l2 mutant mice and
controls. Protein carbonylation is increased in Afg3l2 mutants. Mitochondrial enrichments from brain tissue ( par/par
panel a, Emv66/Emv66 panel b) are treated with DNPH. Derivatized proteins are analyzed by SDS-PAGE on a 10% gel.
Anti-porin and anti-39 kDa antibodies are used to verify equal loading.
loading control. The procedure for secondary antibody
incubation and ECL detection follows exactly the same
instructions described above.
4. Notes
1. Local and national regulations on animal care and handling
vary. It is important to check holding the appropriate autho-
rization to perform animal experiments.
2. All buffers are prepared the same day of the experiment, to
avoid bacterial/yeast growth in stored buffers. Since pH
depends on temperature, measure the pH of all solutions at
25°C.
3. The optimal ratio between tissue and isolation buffer ranges
between 1:5 and 1:10 (w:v).
4. All glassware and dounce have to be washed three times with
bidistilled water to avoid Ca 2+ contamination. Ca 2+ overload is
the most common cause for the dysfunction of isolated
mitochondria.