Hi,

You can find an interesting discussion on this topic here:

Article Mitochondrial Membrane Potential Probes and the Proton Gradi...

I hope that this might be useful for you.

Best regards,

Hi Rachel,

If I have to think in the simplest explanation, I'd say your isolated mitochondria are "uncoupled", therefore you don't see an increase in the respiration rate after adding ADP (I suppose you're adding phosphate and Mg to the reaction mixture). Depending on the method you use to isolate mitochondria, the organelles might be not intact enough and somewhat broken. In order to check this, a control using other substrates might give you an idea if your organelles are still coupled after the preparation; e.g. adding Pyruvate+Malate or Glutamate+Malate to feed electrons via complex I (if this applies on your model) to induce state 2 and then adding ADP (+Pi+Mg) or titrating FCCP (or CCCP) to see if the respiration increases.

Good luck with your experiments! Hope you find a solution. If you need extra help, send me an inbox

Best,

Alfredo