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Product code: 109878
Inquiry: When I assess the complex IV activity will it have retained inhibition from ATP that may or may not be occurring within the samples that I am looking at? Or will it loose this inhibition upon processing? Thanks
Asked on May 14 2012
Thank you or your inquiry.
Please find below the answer of one of our senior scientists to your question:
"Iam assuming that the customer refers to high ATP/ADP ratios reducing the activity of Cytochrome c Oxidase, making it the rate limiting step of the electron transport chain.Kadenbach hypothesized several years ago that inhibition of complex IV by ATP was due to phosphorylation. Since then several researchers have shown that Cytochrome c oxidase is phosphorylated in subunit 1, 4 and 5b by mass spectrometry.
Assuming that the customer is concerned about capturing endogenous CIV activity, then the activity observed with the kit will depend mostly on sample preparation. If the customer is worried about it, my suggestion is to test a control sample with and without phosphatase inhibitors and compare the activity of both preps with the kit. If it is a rodent tissue, care must be taken into account from the moment the animal is euthanized to maintain the “endogenous phosphorylation status “.
We have not tested in the lab the effects of ATP in-vitro on the activity of CIV. But If the customer is interested in determining the ability of ATP to downregulate the activity of CIV after immunocapture, then they will have to adjust the protocol in a way so that they can expose CIV on the dipstick to a titration of ATP. The way I would personally set up the first experiment (although I’ve never done) would be to immunocapture the complex with the dipstick and then remove the wicking pad and dip the dipstick in a contiguous well containing 300uL of buffer with a titration of ATP (including a no-ATP control) for 15 – 30 minutes before developing the dipstick with the activity solution. We have done something similar after immunocapture of PDH and in this case we found that it was necessary to add BSA during the treatment reaction to maintain stability of the immunocaptured complex on the dipstick. PDH is a much larger enzyme that CIV and so even though this is comparing apples and oranges, my point is that the customer should be careful in assessing results when deviating significantly from the protocol."
I hope this information is helpful and wish you good luck with your experiments.
Answered on May 14 2012