I'd like to know if my mice as some impairments in their oxidative balance (in brain).
I'd like to perform biochemical analyses, better than biomolecular.
Dear Guillemette, if you try to measure mitochondrial impairment you could use Seahorse (http://www.seahorsebio.com/applications/neurodegeneration.php). 8-Oxoguanine can be used (by IHC, WB or ELISA) for indirect measurement of the reactive oxygen. Best, Valeria
Please read about TBARS, Catalase, superoxidismutase, GPX activity from brain homogenates.
Do you mean biochemical assays and spectrophotometric measures ?
and for catase activity evaluation chose an UV kinetic method with hydrogen peroxide
Some options:
-Reduced/total glutathione, NAD/NADH, and NADP/NADPH (many kits commercially available, indirect measures of redox status)
-Immunostaining of tissue slices for carbonylated proteins (oxidative damage to proteins), 8-oxo-dG (oxidative damage to DNA), or 4-HNE (oxidative damage to lipids)
-Western blotting for carbonyls
-Kits for measuring carbonyls, 8oxodg, or 4-HNE by chemiluminescent assays
-Metabolome profiling for a more detailed view of redox/antioxidant molecules as well as metabolites from the ETC, TCA cycle, etc.
-Western blotting for protein markers consistent with stress such as Hif-1 (and targets of Hif-1), Nrf2 targets, mtUPR proteins, etc.
-Seahorse using mitochondrial isolates or enzyme activity assays
-Live tissue imaging of fresh tissue sections
Many thanks for all these answers !
I will think about this....
Best to all,
Guillemette
I would suggest to quantify total and reduced glutathione in tissue extract, by using one of the several kits available on the market for GSH assay. GSH accounts for most of total antioxidant capacity inside tissues and an alteration of GSH to GS-SG ratio represents a robust marker of oxidative tress.
Just homogenise the tissue, deproteinize with perchloric acid, centrifuge, neutralise the supernatant with KHCO3, centrifuge and assay glutathione in the supernatant. Carry out sample pretreatment at 0-4°C. The supernatant can be stored at -20°C for several weeks before assaying.
Best wishes,
Gianfranco Liguri
Thank you very much for this answer !
Do you know the minimal quantity of tissue to perform that kind of analyses ?
Best,
Guillemette
An amount as small as 100 mg (or more), homogenized in 1 mL isotonic buffer, would be enough for GSH/GSSG and additional assays. I would suggest, anyway, to start from 500 mg or so,
GL
Dear Guillemette, I regret to say that there is no "easy" way to answer your questions. "Easy" and indirect methods used to prove that there is oxidative stress have added nothing useful to understanding mechanisms and origin of free radicals and their actions. This is because there are so many levels and conditions that affect oxidative stress. First, you have to study mitochondria by direct measurements of respiration and ROS production (Amplex Red method will suffice, but you have to have a really good instrument for measurements of fluorescence of Resorufin). And you have to use right mixtures of substrates that are optimal for your metabolic mouse phenotype.
Glutathion is only one of the players, and not of most importance for the brain. You have to understand that most important damaging effects of the oxidative stress are at the level of synapses.
I would suggest that you read more about ROS in brain tissue and the role of mitochondria. Read recent papers from the Dr. Martin Brown team the Buck Institute. They are fundamental though experiments were made on skeletal muscle mitochondria. As regards brain, I have published recently a number of papers on the isue.
Good luck!
To measure GSH/GSSG do not acidify before blocking free SH groups. Sample acidification in most biological matrices oxidizes GSH to GSSG. You may measure biased values.
Here you can find the suggested procedure, and basal values we found in rat brain for GSH, GSSG and glutathionylated proteins.
http://www.ncbi.nlm.nih.gov/pubmed/23928499
http://www.ncbi.nlm.nih.gov/pubmed/21335026
Protein carbonyl levels are an excellent and sensitive measure of oxidative stress.
see http://www.ncbi.nlm.nih.gov/pubmed/12589963
If you are doing in vivo or histological experiments, the conventional methods would be immunostaining based:
-- 3-nitrotyrosine staining (to assess protein Tyr nitration level)
-- 8-oxo-dG staining (to assess DNA [guanosine] oxidation)
-- MDA ELISA (to assess lipid peroxidation reflected by levels of malondialdehyde)
These are perhaps the best known oxidative stress markers.
GSH-GSSG ratio would be nice, but only if you can make sure no autooxidation occurs to your GSH in samples.
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