Hi all, I am working on cancer cells and my drug shows ROS production and mitochondrial membrane depolarization but I want to check the effect of my drug on mitochondrial respiratory chain complex (MRC) activity. Guide me how to check the MRC activity.
Dear Kunal,
I attached an excellent publication on measurements of respiratory chain activities. In this paper you will find a wealth of information that will help you.
Best regards, Christian
mitochondrial respiratory chain complex (MRC) activity can be assessed using Mitosox, the fluorescent dye reacting with the depolarization reaction in inner membrane of the mitochondria.
Dear Kunal,
I attached an excellent publication on measurements of respiratory chain activities. In this paper you will find a wealth of information that will help you.
Best regards, Christian
The classical activity assays with sub-mitochondrial particles and respiration assays (using different substrates) with isolated mitochondria might be convenient and useful approaches.
You can measure oxygen consumption with Clark electrode, you can measure the production of ATP, you can measure the production of superoxide radical (MitoSOX), you can measure cytochrome oxidase activity, you can use different inhibitors of the different complexes.
Yes, the protocols published in Nature Protocol (article mentioned by Christian Scheckhuber) work. In addition you test your drugs in clear native gels (Electron transport chain complex in-gel assays. Reference: Wittig, Molecular & Cellular Proteomics, 2007, 6:1215-1225.
There is nothing more informative than to study respiratory activities and membrane potential with right selection of substrates for the given organ. To staudy the respiratory complexes separately actually give you no useful information about pharmacological effects of your drug on mitochondria. For this you have to work with submitochondrial particlles. In SMP the activity of Complex II is inhibited and other complexes, particularly complex I, give only relative information about physiology.
I also measured activities of respiratory chain complexes and citrate synthase according to protocol from Spinazzi in Nature Protocols and it worked very well.
O, God!
People, measurements the activities of respiratory complexes has nothing to do with understanding physiology of metabolism and pharmacological effects of the drugs. In fact, even for diagnostic purposes these activities have only limited implications.
There are too many pitfalls in the measurements, therefore what people published is full of errors and misinterpretations. Of course to isolate good mitochondria and study them is much more difficult. I have attached a Chapter from my book describing SMP.
You can use specific inhibitors of specific complexes eg Rotenone, DPI, Myxothiazol etc
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology
- Cancer Cells