Overview

  • Product name

    Complex IV Human Enzyme Activity Dipstick Assay Kit
    See all Complex IV kits
  • Sample type

    Cell culture extracts, Tissue
  • Assay type

    Sandwich (quantitative)
  • Species reactivity

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

    Contains 30 or 90 dipsticks and necessary components to quantify the activity of the cytochrome c oxidase enzyme complex from human and bovine samples. The kit includes sufficient materials to generate a standard curve and evaluate several unknown samples.



    The isolation of mitochondria is not necessary for the performance of this assay. In this kit the specificity of anti-COX monoclonal antibodies (mAbs) is combined with traditional methods for determining COX enzyme activity by histochemical methods and in-gel activity assays. First, the COX enzyme complex is immnocaptured (i.e immunoprecipated in active form) on the dipstick. Second, the dipstick is immersed in COX activity buffer containing reduced cytochrome c and di-amino benzidinetetrachloride (DAB), which serves as the reporter of COX activity. Immunocaptured COX oxidizes cytochrome c, which then oxidizes DAB to form a red-colored precipitate at the COX antibody line on the dipstick. In addition to being quick, the reaction is cyanide-sensitive. The signal intensity of this precipitate corresponds to the level of COX activity in the sample. The signal intensity is best measured by a dipstick reader or may be analyzed by another imaging system.

  • Notes

    Store dipsticks at room temperature in their provided container and out of direct sunlight. High humidity conditions should be avoided.

    Store Buffer A, B, and C at 4°C or at -20°C for long term storage.

    Store Tubes 1 and 2 at -80°C; they can also be aliquoted upon receipt to prevent freeze/thaw cycles.

    Tube 3 can be stored at room temperature.

     

    Range of complex IV / cytochrome c oxidase assay kits

    Biochemical assay - ab239711

    Immunocapture with biochemical assay (plate-based) - ab109911 (rodent)  and ab109909 (human)

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

    Immunocapture with biochemical assay and ELISA - ab109910 (human) 

    ELISA - ab179880 (human)

  • Tested applications

    Suitable for: Functional Studiesmore details
  • Platform

    Reagents

Properties

Applications

Our Abpromise guarantee covers the use of ab109876 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent dilution.

Images

  • Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. Dipstick ELISA Kits extend this concept by utilizing the well-established lateral flow concept, wherein capture antibodies are striped onto nitrocellulose membrane and a wicking pad draws the sample through the antibody bands. 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.

  • Figure 1. An example using ab109876 to measure Complex IV activity in fibroblast protein extracts. Developed dipsticks from a 1:2 dilution series using a positive control sample and the associated standard curve. Starting material was 100 µg of fibroblast protein extract.

  • Figure 2. An example using ab109876 to measure Complex IV activity in fibroblast protein extracts. Based on the standard curve, 50 µg of protein extract were loaded onto a dipstick for each sample. The figure shows four developed dipsticks, a control sample (1) and four unknowns (2-6). The analysis of the signal intensity and interpolation from the standard curve showed that the unknown samples have between 15-61% of normal Complex IV activity levels.

Protocols

References

This product has been referenced in:

  • Li H  et al. Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations. Autophagy 15:113-130 (2019). Read more (PubMed: 30160596) »
  • Rodríguez-García ME  et al. An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene. Hum Genet 136:885-896 (2017). Read more (PubMed: 28526948) »
See all 10 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Answer

Thank you for your inquiry.

I am sorry to confirm that we are not sure what other protein assays will be compatible.

I think Bradford Assay is sensitive to detergents in general and therefore most probably not suitable.

If you like this could be quickly tested by using the extraction buffer alone in the Bradford reagent to see if it changes color.

Another option would be to measure with UV 280 for protein samples. This will also work without any additional material or kits.

I am sorry that I did not have a straight forward answer for you on this occasion and hope this information is nevertheless helpful.

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Answer

Thank you for contacting us.

Cytochrome c is available in our catalog, ab140219

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Answer

Thank you for contacting us.

We don't have exactly what you're looking for unfortunately, but we do have MetaPath™ Mito Disease 4-Plex Dipstick Array, ab109879.

The ab109879 is a novel array that allows for the simultaneous quantification of 4 key enzymes whose expression is down-regulated in different mitochondrial diseases: Complex I, Complex IV, PDH and Frataxin. The array is comprised of 4 monoclonal capture antibodies striped onto a dipstick, and the immunocaptured sample is detected using 4 monoclonal detector antibodies that recognize different epitopes on the target enzymes. This array is very rapid (total assay time including sample prep is less than 1 hour) and very sensitive, and as with all Abcam's assays, isolation of mitochondria is not required.

This array is suitable for testing a variety of sample types. Not only is it useful for measuring samples that are deficient in the 4 enzymes due to genetic mutations, it is also useful for measuring changes in the expression of the enzymes due to drug treatments or other manipulations.

https://www.abcam.com/MetaPath™-Mito-Disease-4-Plex-Dipstick-Array-ab109879.html

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

I'm glad you have found the information helpful. If you do have any further questions please do let me know.

Until then I wish you all the best and good luck with the money-keeper :)

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Answer

I now have further information to share with you in regards to the kits Complex IV human enzyme activity microplate assay kit (ab109909)andComplex IV human specific activity microplate assay kit (ab109910).

As with the Complex IV human enzyme dipstick (ab109876) which you were interested in, these kits are also compatible with the sort of sample you are hoping to use. You could make a pellet of cells and freeze them at -80C as a dry pellet. There is in fact no need to freeze the cells viable.

What we recommend is to do the activity assay on the day the cells are treated with detergent, otherwise they may run the risk of degradation of the sample while stored in the provided detergent at -80C.

I hope this information has been of help. If you have any further information please do not hesitate to contact us again.

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Answer

Thank you for contacting us and your interest in our products.

The Complex IV Human Enzyme Activity Dipstick Assay Kit (ab109876)was validated by MitoScienceswith frozen cell pellets (non viable) from patients with SIRF1 and COX10 mutations leading to complex IV deficiency. The attached paper shows the validation data. Your samples should work well with the kitsince the PBMCs were frozen viable.In order to use them with the kit you would first need tothaw them, wash well the FBS with PBS by centrifugation, then solubilize the pellet according to the protocol instructions and measure protein concentration either by 280nm (UV) or by BCA assay before loading on the dipstick.

The paper attached also shows accuracy with the complex I activity dipstick.

I hope this information has been of help. If you have any further questions please do not hesitate to contact us again.

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Answer

Thank you for contacting us.
Yes these samples can be used for Western blotting just as one would with RIPA, the extraction detergent in A is very similar to RIPA (NP40 plus others) but it is more native.
The only thing that I would say is that when used concentrated and diluted in the Western blot sample buffer, detergent may affect SDSPAGE migration at the very bottom of the lane (much less than 10kDa) but all other migration should be normal.
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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Question
Answer

Thank you for your phone call.

I am sorry this product did not contain buffer Bas stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question
Answer

Thank you for contacting Abcam

I sent you query to our lab to ask their opinion on your data and I have attached the file edited with whatthey consider are the positive peaks (without looking at the actual dipsticks). The positive peaks are highlighted in green. The signal is very weak, but the ones highlighted in green are the only ones that show increase in activity with higher loading. The dipstick reader will allocate number of peaks. The peak of interest will not always appear in the same number. Whatyou should look for is the peak at a particular position of X. In this case, they need to look at the peak between 460 and 490 (which in some dipsticks is peak 1, in others is peak 2 and in others is peak 3).

When loading blood, we typically don’t load it by the protein concentration, since most of it is going to be hemoglobin present in red blood cells and albumin present in the serum which are not representative of the mitochondria content in blood (present in PBMCs and platelets).You should load by uL of extract (and not by ug). In the graph below, we have the data you should expect by loading 90uL of blood extract, 45uL……in a 1:2 dilution series. The data was obtained with pooled blood from 3 healthy volunteers. Up to 100uL of volume could be loaded on the dipstick. The 90uL loading was achieved by combining 90uL of extract with 10uL of 10x blocking.

For optimal results, it is also important for you to use fresh blood (not frozen) or if this is not possible, blood should at the very least be stored at -80C viable by adding 10% DMSO just like you would on cultured cells). We have only tested the dipsticks with fresh blood and so cannot guarantee their performance on frozen viable blood, but theoretically it should work well. Also for higher quality data it is recommended to isolate PBMCs from the blood and use that in the assay as opposed to whole blood samples.

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Answer

Thank you for contacting Abcam regarding this issue.

On occasion the reader will see more profiles which cannot be seeing with the naked eye on the dipstick. This is due sometimes to imperfections of the nitrocellulose or to uneven wicking. For activity assays the antibody line is stripped at about 7mm from the nitrocellulose edge of the dipstick (or lower end of the dipstick). In new readers, the dipstick is inserted with the wicking pad towards the door of the instrument and the nitrocellulose towards the inside of the instrument. The reader will read from the inside of the instrument (lower section of the dipstick) to the outside of the instrument (upper section of the dipstick). Therefore the lower section of the dipstick will appear towards the left of the absorption profile. The reader will pick up all peaks in a sample .



We strongly recommend creating a standard curve using 7 to 8 dipsticks for covering the working range. In this way you will be able to know which peaks are background and which are signal. Following the protein concentration ranges as defined in Table 1 of the protocol, generate a standard curve using a positive control sample. We recommend performing a 1:2 serial dilution with 1 part Buffer A to 1 part Buffer B in a total volume of 50 µl.



Complex IV dipsticks may sometimes develop a percipitate about 2mm from the edge, this should be ignored.



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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1-10 of 11 Abreviews or Q&A

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