Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)

Thank you for contacting us. The lab informed me that sample extracts can be frozen at -80oC when undiluted. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

My guess is that the levels of OXPHOS proteins in whole blood are below the level of detection for activity assays. The activity assays for the most part require higher loading than the sandwich ELISA assays. We have tested Complex IV Quantity with whole blood with good results.



The caveat is that whole blood must be fresh or must be frozen viable (protocols can be found in the literature). Whole blood should not be loaded in terms of mg of protein as most of the protein present in whole blood is albumin, so using the loading guidelines for tissues to load whole blood will result in loading well below the detection level (OXPHOS proteins are present in leukocytes and platelets only). My suggestion for the customer (if they want to use the quantity dipstick kits) would be to mix 300uL of blood with 900uL of extraction buffer, follow the protocol in regards to the rest of the sample preparation (ice incubation, centrifugation, keeping the supernatant and discarding the pellet). I would advise to the customer to add protease inhibitors to the extraction buffer to prevent degradation of proteins after extraction as granulocytes from blood have high levels of proteases, which will be released once the detergent is added.



For loading on the dipsticks, the blocking buffer should be mixed with 50uL – 100uL of extracted blood so that the final concentration of blocking is 1X. This mixture can then be used to resuspend the gold conjugated antibody dried at the bottom of the well. Once the gold is resuspended the dipstick can be added to the well and allowed to wick the protein fully. Results will not be perfectly accurate (as loading cannot be done in terms of mg of protein), but will be more qualitative in terms of percent of signal from control mean or relative units. This approach will therefore require you to set up a control sample, which should be a pooled sample from several normal donors (10 – 20 donors would be ideal). From our experience, whole blood will not be as accurate as measuring isolated PBMCs only. But it will allow you to have a rough estimate of the trend.

The advantage of isolating PBMCs is that they could run both activity and quantity dipsticks/microplates by following the same guidelines of cultured cells.

Thank you for your patience in awaiting our reply. This kit was developed using tissue or cell celtures, and we would expect a higher reading than you got in those types of samples.

Technically no reason why you couldn’t use blood – however I think the concentration of extracellular COX would be very low in serum or plasma even in cases of tissue damage.

If you are anticipating analyzing blood cells I would definitely recommend collecting them and then extracting them as per the protocol.

Please let me know if you have any further questions.

Thank you for contacting us.


Technically no reason why you couldn’t – however I think the concentration of extracellular COX would be very low in serum or plasma even in cases of tissue damage.

If you are anticipating analyzing blood cells I would definitely recommend collecting them and then extracting them as per the protocol,


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us.
We following kits available for complex III. DO you think this the one you are looking for;
https://www.abcam.com/mitotox™-complex-iii-oxphos-activity-microplate-assay-ab109905.html
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you very much for having patience!

I looked at the data -the curve lost linearity after 50 sec because the substrate presumably is starting to deplete (there is less reduced cytochrome c as the enzyme clearly is using it). A way around the issue, is to work only with the data before 50 sec, which from the graph looks very linear.

I hope this information will be helpful. Should you have any question please do not hesitate to contact me.

Thank you very much for your email.

I can confirm that I am still working on your inquiry. I will send you the solution soon.

Thank you very much for having patience!

Thank you for contacting us.
In order to help you with your question we need more information about the protocol used. Please submit any raw data you have.

We will look forward to hearing from you soon.