I am running an assay for mitochondrial complex III activity using isolated mitochondria. I am using antimycin A dissolved in DMSO as an inhibitor bur the antimycin a is not inhibitory to the reaction even at 100 micro M concentration. The assay is based on absorbance of cytochrome C at 550nm. What could be the possible reason for Antimycin A to not inhibit the reaction?
Dear Bahl,
Antimicyn A is better dissolved in Ethanol...
I suggest 2,5 microM as final concentration to the reaction.
- Do you use KCN in your assay cuvette to prevent oxidation of cytochrome c by complex IV?
- Add 20 µl of EDTA (5mM, pH 7.5) to 980 µl assay solution to increase the sensitivity of the measurement.
- Do you solubilize the mitochondria by using a detergent like digitonin?
- Are the complex III activities you measure reasonably high?
- The publication in the attachment might give you some valuable hints how to solve your problem.
Good luck and regards, Christian
Article Assessment of mitochondrial respiratory chain enzymatic acti...
Hi Christian,
Thanks for your suggestions. I added sodium azide to block oxidation of cytochrome c by complex IV, although I did not add EDTA or detergent. I will try using those.
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