Question (34055) | MitoTox™ Complex V OXPHOS Activity Assay Kit (ab109907)

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Question

Hi Abcam, I ordered the MitoTox™ Complex V OXPHOS Activity Microplate Assay (ab109907) before. It works great. However, I needed to include a "negative control" in my work specifically, I wanted to have a compound that does not effect the activity of the complex (since all of the compounds I tested actually decreased the activity of the complex). Do you have a suggestion for a negative control? (Don't say water or ethanol). Thank you Abcam!

Answer

Thank you for your enquiry.

The negative control chosen for ab109907 MitoTox™ Complex V OXPHOS Activity Microplate Assaywould depend on whatyou want to accomplish and obtain from the experiment.

Some people test the specificity of the assay by testing specific and non-specific inhibitors of the electron transport chain. For example, you could test for MTOX5 oligomycin as positive control andantimycin as negative control (which is specific inhibitor for complex III and will not inhibit complex V). The dose for the negative control is important,our suggestion is 10 uM.

However if the interest is not specificity but simply a compound that has no effect in any of the complexes, but it is known to affect mitochondria by a different mechanism then we would suggest using chloramphenicol (10 – 50 uM).Thisshould not affect any of the MTOX assay kits.

There are many others we could suggest, but without knowing exactly what the aim of your negative control is, it is difficult to do so.

Other options could be dinitrophenol or FCCP. In an invitro system like the MTOX assays DNP and FCCP will not affect the activity of the enzymes. However, in a cultured system DNP and FCCP will uncouple mitochondria.

I hope this will be helpful. if you require any further information in this case, please do not hesitate to contact me again with further details of thetype of negative control you require and we can investigate this further for you.

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