The mitochondria is the primary target for oxidative stress induced by Cisplatin, resulting in loss of mitochondrial protein sulfhydryl group. We have some interesting data on Oxidative stress biomarkers - CAT, SOD and GPX. But, I need some help in estimating the mitochondrial protein level for better conclusion. ANy suggestions please ?
Hi Shaloam,
MitoSciences offers several kits to assess mitochondrial abundance and activity. This kit: http://www.abcam.com/Complex-I-Human-Protein-Quantity-Dipstick-Assay-Kit-ab109722.html will allow you to quantify mitochondrial complex I, which can be an appropriate proxy for total mitochondrial protein content. You can browse around their website for other options too.
Alternatively, you can perform a Western blot for TOM20 or other mitochondrial markers as a proxy for mitochondrial protein abundance.
If you have a fluorescence microscope and analysis software that can quantify fluorescence signals, you can also stain your cells with MitoTracker Green and quantify the signal as a measurement of total mitochondria.
I have used each of these in the past with good success. All of these approaches are pretty straight forward and routinely accepted in the literature as indicators of mitochondrial abundance.
I agree with Nathan that there are several good options for normalization. You could also think about measuring citrate synthase (CS) activity to normalize your data. There are CS assay kits available from Sigma-Aldrich and Abcam, for example.
Best regards, Christian
Are you working with isolated mitochondria? If you are working with isolated mitochondria, you can use simple protein estimation methods (Bradford. Lowry etc.). Although, I do partially agree with Nathan, I feel that it is an indirect method. If you isolate intact mitochondria and determine mitochondrial SOD and GPX, I will prefer direct protein estimation using simple protein quantifications methods. Of course, CAT activity will not be seen in pure mitochondrial preparation, however, if it is contaminated with microbodies (which is generally is the case), then you can see catalase activity as well. This is just a thought and I may be wrong.
Greetings!
Dr. Pradha-Saradhi recommended Bradford method. Very simple and reliable. But, in order to understand what you do, I would like to know what is the Object of your study: what animal, organ or cells?
From my own experience I know that one has to be very careful with quantification based on Western blots. You have to have some interbal calibration. Can you give more information?
As regards p[rotein determination in isolated mitochondria, Bradford works fine. However, there are some tricks, which I have described in my book "Practical Mitochondriology". You can can copy it from my site on ResearchGate, or buy printed version via Amazon.
Sincerely,
Alexander Panov
Thank you Dr. Pardhasaradhi and Dr. Alexander for your valuable suggestions. I work with HL 60 cells (Acute promyelocytic leukemia ).
There are several possibilities to normalize your enzyme activities. If you isolate your mitochondria, you also can measure your protein level by Bradford assay, easy and reliable. But if you want to compare respiratory chain complex activity you should normalize it to a certain protein. I suggest, that citrat synthase is best for normalization. It should not be much affected by any treatment. And you also can use "older" methods for the determination of CS aactivity and you have not to switch to expensive kits.
Best
As regards submitochondrial particles and normalization on activity of CS< I would recommend to read some of the recommendations from my book Practical mitochondriology. Here, I have attached a PDF pf the Chapter 5 from that book.
Normalization does not work. Rustin was incorrect..
You can find out specific activity of marker enzyme, say succinate dehydrogenase in isolated mitochondria. Then go back and do the same with homogenate/your system/cell culture. Based on this you can back-calculate the content of mitochondria in the given syste. No kits are required. e.g. see our attached PDF
SDH is, in fact, one of the most difficult enzymes to study. The enzyme eaily becomes inactivated during sonication (preparation of SMP), and is the subject to significant variations in activity due to endogenous inhibition by oxaloacetate. In addition, any changes in energization of mitochondria affect this inhibition.
You do not have to sonocate. Just use whole mitochondria, PMS and DCIP. Monitor decrease in absorbance at 660 nm and that is all. It is all hassle free. The enzyme is quite stable in whole mitochondria..
Thanks for the materials Dr. Alexander and Dr. Surendra. Yes, I use Pencilin in my media at a concentration of 5ml/500ml of IMDM.
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