Complex I Enzyme Activity Dipstick Assay Kit (ab109720)

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Samples prepared for dipstick can also work in microplate and vice vera. You could reference to the following guide for sample preparation:

http://www.mitosciences.com/PDF/mitosciences-sample-preparation-guide.pdf

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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In regard to your inquiry please find the following comments:

1. Would I use MCF7 cells treated with rotenone?

The treated MCF7 cells should be used exclusively as test samples and not as standard curve. Bear in mind that if you treat MCF7 cells with rotenone, then wash, trypsonize and harvest for generating a lysate it will be highly unlikely that you will see a decrease level of activity of complex I. there are two reasons for this. One, by the time the sample is loaded the drug will have been washed away. Two, the dipstick kit measures the dehydrogenase part of the enzyme only and it does not measure the full activity. Therefore it is not rotenone sensitive. This is explained in detail in the Background section of the protocol. If the customer wants a kit that is sensitive to rotenone, I suggest to use https://www.abcam.com/mitotoxtrade-complex-i-oxphos-activity-microplate-assay-ab109903.html

2. Do I establish the protein concentration of the positive control and do a titration to establish a standard curve of activity? I'm a little confused on the positive control and standard curve part.

Yes, you need to determine protein concentration with a standard protein quantitation kit like the BCA assay. Once you know the concentration of your lysate, you will be able to load on each dipstick based on the typical sample dynamic range table (page 9 of the protocol). For MCF7 cells I suggest to start in the same range of Fibroblast extract. To minimize the use of dipsticks in the first test, I suggest to load control MCF7 at 30ug/dipstick, 10ug/dipstick and 3ug/dipstick. Once it is developed, you will have to make a judgment call on whether the dynamic range should start higher or lower depending on the results. If the signal does not titrate between 30 and 10, then the dynamic range should start lower. If there is no signal at 10ug/dipstick, then the curve should start higher.

I hope this information adequately covers your questions. Please feel free to contact me for futrher assistance. Take care.

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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Cytochrome c is available in our catalog, ab140219

Thank you very much for your email and for your interest in this dipstick figure.

I've forwarded your request to our commercial team, and we will be in touch shortly.

Please let me know if you have any questions, and thank you for your patience.

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With regards to ab109720, there is a publication on the utility of Complex I enzyme activity dipsticks in patients with specific mutations in the different subunits of complex I. The antibody stripped to the nitrocellulose is capable of capturing the multisubunit complex in its native form when the sample is extracted with the detergent included in the kit. The attached paper shows in detail how the antibodies were generated, how they were validated by mass spectrometry (see reference 14 for complex I) and how the dipstick activity kit performs on fibroblasts from patients with mutations in specific subunits of complex I.

With regards to ab109882 , the capture antibody used in the kit targets E2 – clone ID 15D3G9C11 (ab110332). IP characterization of this antibody was also published. The focused proteomics paper, also attached, shows how all subunits of PDH co-IP when using this antibody (in the paper is referred to as (15D3).

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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We have not tested the complex I dipstick immunocapture activity kit with Triton. Homogenizing the samples mechanically is not enough. This kit was optimized to extract complex I into its native form (with all 47 subunits assembled in to a large complex). This is a pre-requisite for the capture of an active complex I on the dipstick. We don’t know whether Triton inactivates complex I or not or whether it can extract it into its native form. My suggestion would be to use the detergent provided with the kit on fresh PBMCs or a frozen pellet of PBMCs (kept at -80C), extract with addition of a protease inhibitor cocktail and perform the assay on the day of sample extraction. We do not recommend performing the assay on frozen lysates, particularly from PBMCs.
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 have contacted the laboratory. They confirmed to me thatthey have seen this problem before with viscous samples.

In order to determine more the problem obtained in your case, can you please answer the following questions? Thank you very much.

What type of sample are you using?The laboratory has noticed this kind of problemin particular with blood samples (this is why we do not recommend this sample) as well as with degraded samples or samples that need to be loaded at a very high concentration.
Are the original lysates viscous? Or can they easily be resuspended?
At what loading concentration or quantity areyou wicking the samples on the dipstick?
What volume do youwick? Do you wick 25uL sample in buffer a + 25uL of of buffer B or do you wick 50 + 50?
Have youfound this to be an issue with a particular lot of dipsticks? Or do all dipstick lots behave the same?



You are right in that differences in wicking speed will change the result. The only one that can be trusted is the dipstick which wicked at a similar speed to the calibration curve.

Sinceyou are using an activity kit, the suggestion of the laboratory for now would be to wick the samples in separate wells rather than on contiguous wells. For example dipsticks should be wicked with a column empty in between (A1, A3…) and 3 rows empty in between (A1, E1). This arrangement will allow more air between the dipsticks and it will give a more even wicking. The closer together the dipsticks are (particularly on difficult samples that need to be run in a batch analysis) the more uneven the wicking will be.My contact in the laboratory haspersonally wicked 48 samples on a single plate (whichshe would however not recommend) with no issues using non viscous samples.Shehas also tested 96 in one plate (which againshe does not recommend) and has found that the dipsticks in the center of the plate wick much slower.

I hope this information is helpful already. We look forward to hear back from you.

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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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