Overview

  • Product name

    Complex IV Human Enzyme Activity Microplate Assay Kit
    See all Complex IV kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture extracts, Tissue
  • Assay type

    Enzyme activity
  • Species reactivity

    Reacts with: Cow, Human, Pig
    Does not react with: Mouse, Rat
  • Product overview

    The Complex IV Human Enzyme Activity Microplate Assay Kit is used to determine the activity of cytochrome c oxidase in a human sample with greater speed and simplicity. The COX enzyme is immunocaptured within the wells of the microplate and activity is determined colorimetrically by following the oxidation of reduced cytochrome c by the absorbance change at 550 nm. Included in this kit for performance of the activity assay are buffer, detergent, substrate, and 96-well microplate with monoclonal antibody pre-bound to the wells of the plate, allowing for a stream-lined assay.


    Store Tube 1, detergent, and microplate at 4°C – DO NOT FREEZE. Store Reagent C at -20°C or at -80°C for long-term storage.

  • Notes

    Range of complex IV / cytochrome c oxidase assay kits

    Biochemical assay - ab239711

    Immunocapture with biochemical assay (plate-based)*** - ab109911 (rodent)  and ab109909 (human) (this kit)
    *** Most popular assay format

    Immunocapture with biochemical assay (dipstick) - ab109878 (rodent) and ab109876 (human)

    Immunocapture with biochemical assay and ELISA - ab109910 (human) 

    ELISA - ab179880 (human)


    Other related products

    Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Platform

    Microplate reader

Properties

Images

  • Figure 1. To determine the activity in the sample, calculate the slope by using microplate software or by manual calculations using one of the two methods shown. Compare the sample rate with the rate of the control (normal) sample and with the rate of the null (background) to get the relative Complex IV activity. (A)The rate is determined by calculating the gradient of the initial slope over the linear region. (B) The rate is determined by calculating the slope between two points within the linear region.

    Figure 1. To determine the activity in the sample, calculate the slope by using microplate software or by manual calculations using one of the two methods shown. Compare the sample rate with the rate of the control (normal) sample and with the rate of the null (background) to get the relative Complex IV activity. (A)The rate is determined by calculating the gradient of the initial slope over the linear region. (B) The rate is determined by calculating the slope between two points within the linear region.
  • Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. All of our ELISA kits utilize highly validated monoclonal antibodies and proprietary buffers, which are able to capture even very large enzyme complexes in their fully-intact, functionally-active states.

    Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, substrate is added, and enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed). By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.

Protocols

References

This product has been referenced in:

  • Andreux PA  et al. Mitochondrial function is impaired in the skeletal muscle of pre-frail elderly. Sci Rep 8:8548 (2018). Read more (PubMed: 29867098) »
  • Llobet L  et al. Pharmacologic concentrations of linezolid modify oxidative phosphorylation function and adipocyte secretome. Redox Biol 13:244-254 (2017). Read more (PubMed: 28600981) »
See all 21 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

Answer

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.  

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Complex IV activity in HCT116 cells

Excellent Good 4/5 (Ease of Use)
Abreviews
We cultivated HCT116 cells in DMEM containing high (4,5 g/l) and low (1 g/l) glucose for 48 hrs. Cells were harvested and lysed as described in the manual. We used Bradford assay to quantifiy and adjust the protein concentration in each sample.
The measurement took place at A= 450nm and not at 550nm. This was possible because the spectra of red/ox cytochrome c differ highly at 550nm and 450nm. When using 550nm you would get decreasing kinetics, at 450nm you get increasing kinetics (lower fig.).
The activity of complex IV is higher in HCT116 cells cultivated in low glucose DMEM then in cells cultivated in high glucose DMEM. The absorbance change is related to the applied protein amount, but does not show a linear relation within the range we tested (upper fig.).
In general the kit is easy to use but takes a lot of time to perform. The results seem to be reliable.

Mr. Christian Marx

Verified customer

Submitted Jul 24 2019

complex IV enzyme activity assay kit

Good Average 3/5 (Ease of Use)
Abreviews
this kit was disigned for use of human samples. I was try to use it to test the porcine kidney and myocardio. The good news is it worked for both tissues.

Abcam user community

Verified customer

Submitted Mar 18 2014

Question
Answer

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.

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Question
Answer

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.

Read More

Answer

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.

Read More

Answer

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.

Read More

Answer

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.

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Answer

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!

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1-10 of 17 Abreviews or Q&A

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