Complex IV Rodent Enzyme Activity Dipstick Assay Kit (ab109878)

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.

Thank you for your inquiry.

I can confirm that Dipsticks could be scanned with a regular document scanner and then signal intensity of the image could be analyzed with Image J software (which can be downloaded for Free).

The other option is to simply use visual inspection. This can be done on the dipstick kits that contain a control protein (Frataxin or GFP). The visual inspection method also requires to user to set aside at least 8 dipsticks to generate a calibration curve (with small gaps between data points). The calibration curve generated with the standard protein can be made into a “visual reference card”. The test sample is then compared against the visual reference card by matching up the reporter line density of the processed dipstick in the appropriate slot (see image below). The value (or value range) can then be interpolated based on the position relative to the antigen levels of the two lines immediately to the left and to the right. The accuracy of the value will be inversely proportional to the dilution factor of the calibration curve.

Dipstick Visual Reference Card Interpolation is attached. The unknown test sample reporter line fits neatly into the slot between the standard #6 and 7.

We have tested this last method with the GFP dipstick and the Frataxin dipstick (using 4 – 8 naïve testers) and have obtained a percentage error less than 15% in comparison to the instrument reading.

I hope this information is helpful.

Thank you for contacting us.

The buffers for each kit are optimized for use with the particular assay. The name 'A', 'B', etc. is by convention and not representative of similar components.

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.

Yes, both kits work with frozen mouse tissue samples.

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.  1. The detergent used in this kit is not efficient at extracting nuclear samples.  Most of the nuclear proteins will be in the pellet obtained after the centrifugation step during the sample preparation (as explained in the protocol).  Other membrane and cytoplasmic proteins can be found in the extract obtained with the kit ab109878, however these proteins will be in their native form.  It will be necessary to mix this extract with 2XSDS buffer before running the samples on a western blot.   2. Solution 1 does not contain protease inhibitors.  In the additional materials required section of the protocol we suggest the use of protease inhibitors.   3. My suggestion is to use between 1 and 5mg of wet tissue in 100 - 200uL of buffer during the homogenization.  Protein concentration in the homogenate should be greater than 1mg/mL. With such  a small sample I suggest to use a very small glass dounce homogenizer (0.5mL). 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. Please find further information below about the use of protease inhibitors: When should protease inhibitors be added?   Protease inhibitors should be added during the extraction procedure.  Which protease inhibitors should be used?   We recommend any off the shelf cocktail of protease inhibitor Will EDTA interfere with Complex IV activity?   The role of Magnesium, Zinc and Sodium ions in the activity of cytochrome c oxidase (to my knowledge) is not yet fully understood and therefore the outcome of adding a chelator of this ions is all but a guess.  Cytochrome c oxidase is composed of 13 subunits, 3 of which (I, II and III) form the catalytic core of the enzyme.  The other subunits are involved in assembly, stability and regulation of the complex.  There is Mg bound at the interface of subunits I and II of complex IV.  Zn is bound to subunit Vb.  There is Sodium bound to subunit I at a site which also accepts calcium and this has been shown to affect the spectral properties of the haeme a3 (which is critical for activity). Typically the activity buffer to carry out a CIV activity assay, contains a very small amount of EDTA.  My suggestion is for the customer not to add more than 5 or 10uM EDTA final concentration to avoid any potential artifacts. We will add this to the protocol for other customers, thanks for bringing this to our attention. Please let me know if I can be of further help.