I am running an assay to look for activity of mitochondrial complex 1 in isolated mitochondria. Reaction mix has a pH of 7.4 with 3mM EDTA, 3mM NaN3 and -.3% BSA along with ubiquinone and NADH. in the blank, whjere no mitochondria is added, there is a slight increase in abs at 340 nm over time. Moreover the rxn with mitochondria is no different from blank. what could be the reason for this?
Thanks
Hello, Vasundhara Bahl
About how much is this increase in absorbance that you observe over time? It is small? How much is the time you are reading? If time is long, the NADH may be suffering a slight oxidation process.
If the run with mitochondria is no different from blank maybe the problem is in the isolation of mitochondria.
I hope your answer and that this will be helpful.
Regards,
Carolina
Hi Carolina,
Thanks you for the answer. The increase is very small: about 0.03 over 3 minutes. The oxidation of NADH would form NAD+ and therefore should the absorbance not decrease since NAD+ does not absorb at 340 nm?
AS for the mitochondria, the ones that I am using are showing very consistent Complex III activity. Could it be possible that Complex I is not stable due to its large size?
Thank you
Hello Vasundhra,
OK, 3 minutes is not a very long time, and neither 0.03 is a great increase in absorbance. I try to think why is that mitochondria give no differences with the blank. I could not tell if the complex I is not stable but I would suggest that you try to check the mitochondria isolation protocol. I hope you can find out where the problem is and I remain at your disposal.
Regards,
Carolina
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