Complex IV Human Enzyme Activity Dipstick Assay Kit (ab109876)

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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Cytochrome c is available in our catalog, ab140219

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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.

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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 :)

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.

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.

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.

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.

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.

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.