Hi Vasundhra,

The azide (or more typically KCN) is in this assay to prevent oxidation of the electron acceptor (most relevant when cytochrome c is the acceptor, but it's often used in assays when exogenous quinone is the acceptor as well). Two thoughts for your assay -(1) Make sure your NADH has been made up and stored correctly, it needs to be reduced for this assay to work, and (2) If your mitochondria have been frozen and then thawed, you want to make sure you have added the electron acceptor in excess because the loss of membrane integrity usually results in endogenous cytochrome c loss. Hope this helps.

Best of luck in your research,

Casey

Hi Casey,

Thank you so much for your insight. As for NADH, I assume it is the reduced form and can be prepared as  a solution in water and then stored at -20 deg C. Is there any additional step I can incorporate to make sure it stays in the reduced form?

Regards.

Hi again,

Water is not the best option because it tends to be acidic and can contribute to the decomposition of NADH. My recommendation is to make it up in 0.1 M NaOH or Tris with a pH in the 8.5-11 range. Aliquot and store in dark tubes at -80, and do not freeze-thaw the solution more than once or twice at the most. This should generate a very stable NADH solution - it lasts a long time in the freezer if prepared this way. 

-Casey

Thanks Casey,

I will try this. I appreciate your help.