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    products/assay-kits/complex-iv-human-enzyme-activity-microplate-assay-kit-ab109909.pdf

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Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)

  • Datasheet
  • Protocol Booklet
Reviews (2)Q&A (15)References (42)

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- Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)
  • Functional Studies - Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Sample type: Cell culture extracts, Tissue

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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

  • Storage instructions

    Please refer to protocols.
  • Components 96 tests
    96-well microplate (12 strips) 1 unit
    Tube 1 (Buffer) 1 x 10ml
    Detergent 1 x 1ml
    Reagent C (Reduced Cytochrome c) 1 x 1ml
  • Research areas

    • Signal Transduction
    • Metabolism
    • Mitochondrial
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Other Metabolism Assay
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Mitochondrial markers
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Cytochromes
  • Alternative names

    • cco
    • COX
    • Cytochrome c oxidase
    • Cytochrome oxidase
  • Database links

    • Entrez Gene: 4512 Human
    • Entrez Gene: 4513 Human
    • Entrez Gene: 4514 Human
    • Entrez Gene: 1345 Human
    • Entrez Gene: 1351 Human
    • Entrez Gene: 1329 Human
    • Entrez Gene: 1349 Human
    • Entrez Gene: 1337 Human
    • Entrez Gene: 1327 Human
    • Entrez Gene: 1340 Human
    • Entrez Gene: 1350 Human
    • Entrez Gene: 9377 Human
    • Entrez Gene: 1346 Human
    • SwissProt: P00395 Human
    • SwissProt: P00403 Human
    • SwissProt: P00414 Human
    • SwissProt: P13073 Human
    • SwissProt: P12074 Human
    • SwissProt: P15954 Human
    • SwissProt: P24311 Human
    • SwissProt: P10176 Human
    • SwissProt: P14854 Human
    • SwissProt: P09669 Human
    • SwissProt: P24310 Human
    • SwissProt: P20674 Human
    • SwissProt: P10606 Human
    see all

Associated products

  • Positive Controls

    • Bovine Heart Mitochondria (ab110338)
  • Related Products

    • Complex IV Human Specific Activity Microplate Assay Kit (ab109910)

Images

  • - Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)
    - Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)
    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.
  • Functional Studies - Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)
    Functional Studies - Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909)
    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

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • Datasheet download

    Download

References (42)

Publishing research using ab109909? Please let us know so that we can cite the reference in this datasheet.

ab109909 has been referenced in 42 publications.

  • Rajab BS  et al. Differential remodelling of mitochondrial subpopulations and mitochondrial dysfunction are a feature of early stage diabetes. Sci Rep 12:978 (2022). PubMed: 35046471
  • Zhu Z  et al. FKBP4 regulates 5-fluorouracil sensitivity in colon cancer by controlling mitochondrial respiration. Life Sci Alliance 5:N/A (2022). PubMed: 35981890
  • Acoba MG  et al. The mitochondrial carrier SFXN1 is critical for complex III integrity and cellular metabolism. Cell Rep 34:108869 (2021). PubMed: 33730581
  • Li J  et al. Association of Mitochondrial Biogenesis With Variable Penetrance of Schizophrenia. JAMA Psychiatry 78:911-921 (2021). PubMed: 34009292
  • Ramchandani D  et al. Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis. Nat Commun 12:7311 (2021). PubMed: 34911956
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 17 Abreviews or Q&A

Complex IV activity in HCT116 cells

Excellent Good 4/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Mr. Christian Marx

Verified customer

Submitted Jul 24 2019

complex IV enzyme activity assay kit

Good Average 3/5 (Ease of Use)
Abreviews
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Mar 18 2014

Question

at what temp. can samples be stored after preparation, before use in the assay kit? can they be frozen at -20 degree for overnight storage? samples are a human cultured cell line

Read More

Abcam community

Verified customer

Asked on Nov 15 2011

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.  

Read More

Abcam Scientific Support

Answered on Nov 15 2011

Question

Do you have any recommendations for OXPHOS assays made specifically for blood?

Read More

Abcam community

Verified customer

Asked on May 24 2013

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.

Read More

Abcam Scientific Support

Answered on May 24 2013

Question

my reading is 0.003, is this low? Is there a known range?

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Abcam community

Verified customer

Asked on Apr 18 2013

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

Abcam Scientific Support

Answered on Apr 18 2013

Question

Hi,



For Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909) can I use blood as sample instead of tissue/cell culture extracts?

Read More

Abcam community

Verified customer

Asked on Mar 28 2013

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

Abcam Scientific Support

Answered on Mar 28 2013

Question

I’ve used the Microplate Enzyme Activity ELISA Kits in your catalogue (ab109721 and ab109909) to measure complex I and complex IV activities with brilliant results, I was wondering whether you provided the same type of kit for complex III or complex II+III activity measurements as well?

I can’t find anything like this on your catalogue but I’d be really interested if you had any alternative.

Thanks a lot for our help,

Best

Read More

Abcam community

Verified customer

Asked on Jan 16 2013

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

Abcam Scientific Support

Answered on Jan 16 2013

Question

Hello and thank you for your response. I have attached an excel file showing data for one of my samples.

The protocol I used was as per manual, product number ab109910.

The assay was done at a concentration of 0.5ug/ml, which us equal to 100ug of protein per well.

Many thanks,

Read More

Abcam community

Verified customer

Asked on Nov 15 2012

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.

Read More

Abcam Scientific Support

Answered on Nov 15 2012

Question

Hello and thank you for your response. I have attached an excel file showing data for one of my samples.

The protocol I used was as per manual, product number ab109910.

The assay was done at a concentration of 0.5ug/ml, which us equal to 100ug of protein per well.

Many thanks,

Read More

Abcam community

Verified customer

Asked on Nov 14 2012

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!

Read More

Abcam Scientific Support

Answered on Nov 14 2012

Question

Hello,

I am using the Complex IV Human Enzyme Activity Microplate Assay Kit ab109909.

As far as I am aware Complex IV has a first order reaction rate, which means that I convert the Absorbance to the natural log of Absorbance ln(A) and plot that against time, I should expect to see a straight line.

I have just tried doing this with my data, and it seems that it doesn’t follow the 1st order reaction rate. I am wondering why that could be? Does this have to do with the optimization of the kit?

Thank you in advance for your help.

Read More

Abcam community

Verified customer

Asked on Nov 05 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Nov 05 2012

1-10 of 17 Abreviews or Q&A

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