In Vivo Detection of Oxidized Proteins: A Practical Approach to Tissue-Derived Mitochondria - Protein Misfolding and Cellular Stress in Disease and Aging - page 255

5. Homogenization, as well as the following steps, must be
performed at 4°C to minimize activation of damaging phos-
pholipases and proteases. Dounce should be precooled in an
ice-bath 5 min before starting the procedure.
6. The minimum volume of buffer should be used to resuspend
mitochondria. The protein concentration should be between
5 and 10 mg/ml. Avoid diluting mitochondria as they retain
their functionality for a longer time when kept concentrated,
minimizing exposure to oxygen.
7. Perform the derivatization of mitochondrial proteins within
1-3 h after mitochondrial preparation. Avoid freezing mito-
chondrial preparation and perform derivatization later. This
will help preventing sample oxidation.
8. For protein quantification, the mitochondrial lysate must be
diluted in order to avoid saturation of the probe.
9. DNPH is very toxic and harmful if swallowed or inhaled;
avoid contact with skin and eyes, wear gloves and eye protec-
tion, use under chemical hood.
10. Generally, 20-50 mg of protein is recommended per derivati-
zation reaction.
11. Avoid high protein concentration (>10 mg/ml) to assure solu-
bility during the derivatization reaction.
12. Do not allow the derivatization reaction to proceed more
than 15 min, as side reactions other than hydrazone linkage
may occur. This also prevents the effect of acid hydrolysis of
proteins.
13. Some protocols suggest the addition of SDS (1-5% w/v) to
protein samples to increase the efficiency of DNPH reaction.
We have observed no differences in the efficiency of derivati-
zation between protein samples treated with or without SDS.
14. Do not heat samples prior to loading into the gel. This pre-
caution preserves the stability of the derivatized residues.
15. Check that remaining gel in Falcon tube is polymerized before
proceeding.
16. It is important to wear gloves when handling membranes to
prevent contamination.
17. Ponceau S staining is a reliable way of measuring total pro-
teins transferred to the membrane and to demonstrate equal
loading. Anyway, given the importance of the quantitative
comparison in this experiment, more sensitive methods to
check protein loading (immunoblot using antibodies against
housekeeping proteins or Silver staining) are recommended.
18. Perform Ponceau S staining before blocking to avoid interfer-
ence with the subsequent antibody reaction with derivatized
proteins.