Complex I Enzyme Activity Dipstick Assay Kit (ab109720)

Reviews (1)Q&A (21)References (48)
Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit - 48 tests (ab109720)
  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)

Overview

  • Product name

    Complex I Enzyme Activity Dipstick Assay Kit
    See all Complex I kits

  • Sample type

    Cell culture supernatant, Cell culture extracts, Tissue

  • Assay type

    Enzyme activity

  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human

  • Product overview

    Contains 48 dipsticks and necessary components to quantify the activity of the Complex I enzyme complex in human, bovine, rat and mouse samples. The kit includes sufficient materials to generate a standard curve and evaluate several unknown samples.
    In this assay the specificity of anti-Complex I monoclonal antibodies (mAbs) is combined with the well-characterized Complex I in-gel activity assay that is not rotenone sensitive. First, Complex I is immunocaptured (i.e. immuno-precipitated in active form) on the dipstick. Second, the dipstick is immersed in Complex I activity buffer solution containing NADH as a substrate and nitrotetrazolium blue (NBT) as the electron acceptor. Immunocaptured Complex I oxidizes NADH and the resulting H+ reduces NBT to form a blue-purple precipitate at the Complex I antibody line on the dipstick. The signal intensity of this precipitate corresponds to the level of Complex I enzyme activity in the sample. Combined with dipstick assay kit for measuring Complex I quantity (ab109722/MS131 for human sample; ab109875/MS133 for rodent samples), it is possible to determine the relative specific activity of immunocaptured Complex I. The signal intensity is best measured by a dipstick reader or may be analyzed by a standard imaging system.

  • Tested applications

    Suitable for: Functional Studiesmore details

  • Platform

    Reagents

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab109720 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.
Notes
Functional Studies
Use at an assay dependent dilution.

Images

  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
    Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)

    An example using ab109720 to measure Complex I activity in human fibroblast samples. Developed dipsticks from a 1:2 dilution series using a positive control sample and the associated standard curve. Starting material was 30 µg of fibroblast protein extract.

  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
    Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)

    An example using ab109720 to measure Complex I activity in human fibroblast samples. Based on the standard curve, 15 µg of protein extract were loaded onto a dipstick for each sample. The figure shows four developed dipsticks, a control sample (1) and three unknowns (2-4). The analysis of the signal intensity and interpolation from the standard curve showed that the unknown samples have between 14-61% of normal Complex I activity levels.

  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit - 48 tests (ab109720)
    Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit - 48 tests (ab109720)
    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.
  • Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
    Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)This image is courtesy of an Abreview submitted by Christian Marx

    ab109720 was used to quantify complex I activity in HCT116 cells. The kit was used as described in the manual.

    Shortly: cells were scraped and lysed in extraction buffer. Cell fragments were removed bey centrifugation and protein concentration was measured by Bradford assay. Increasing amounts of protein (as shown in image) were applied to each well as standard curve. A standard flatbed scanner was used instead of a Dipstick-reader. Due to the low contrast of the resulting bands, the brightness and contrast of the image was adjusted afterwards. Developed for 45 minutes as recommended by the manual.

References (48)

Publishing research using ab109720? Please let us know so that we can cite the reference in this datasheet.

ab109720 has been referenced in 48 publications.

  • Torraco A  et al. A novel homozygous variant in COX5A causes an attenuated phenotype with failure to thrive, lactic acidosis, hypoglycemia, and short stature. Clin Genet 102:56-60 (2022). PubMed: 35246835
  • Suárez-Rivero JM  et al. UPRmt activation improves pathological alterations in cellular models of mitochondrial diseases. Orphanet J Rare Dis 17:204 (2022). PubMed: 35581596
  • Álvarez-Córdoba M  et al. Therapeutic approach with commercial supplements for pantothenate kinase-associated neurodegeneration with residual PANK2 expression levels. Orphanet J Rare Dis 17:311 (2022). PubMed: 35945593
  • Archer SL  et al. SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia. Redox Biol 58:102508 (2022). PubMed: 36334378
  • Álvarez-Córdoba M  et al. Down regulation of the expression of mitochondrial phosphopantetheinyl-proteins in pantothenate kinase-associated neurodegeneration: pathophysiological consequences and therapeutic perspectives. Orphanet J Rare Dis 16:201 (2021). PubMed: 33952316
View all Publications for this product

Customer reviews and Q&As

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1-10 of 22 Abreviews or Q&A

Complex I activity assay in HCT116 cells

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
We used this assay kit to quantify complex I activity in HCT116 cells. The kit was used as described in the manual.
Shortly: cells were scraped and lysed in extraction buffer. Cell fragments were removed bey centrifugation and protein concentration was measured by Bradford assay. We applied increasing amounts of protein (fig.) to each well as standard curve.
In general the kit is easy to use and gives results within 4 h. Although a colorimetric assay would be more precise.
Some problems may occur during signal evaluation. The manual recommends a Dipstick-reader to quantify signal intensities. We used a standard flatbed scanner instead. But due to the low contrast of the resulting bands, we had to adjust brightness and contrast of the pictures afterwards. A Dipstick-reader would probably be the better choice. Another solution for this problem can be to develop the sticks for a longer time span and to increase signal intensity. I developed them for 45 min, as recommended by the manual.
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 Nov 15 2013

Question

Thanks for your last reply!

I have another question. I read the protocols of both kits (109721 and 109720). For preparing tissue samples, in dipstick kit, there are procedures about how to prepare, while in microplate kit, there is no relative procedure indicating how much tissue to start with and which buffer to use to homogenize the tissue.

Can you explain more of those questions?

Thank you very much!

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Answer

Thank you for contacting us.

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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Question



Inquiry: I'm going to be using Complex I Enzyme Activity Dipstick Assay kit. I'm going to be using MCF7 breast cancer cells after treatment of a suspected Complex I inhibitor. I plan on doing a Bradford assay to quantitate protein levels in my samples. On page 12 of the manual it says, "Following the protein concentration ranges as defined in typical sample dynamic range table in section 10, generate a standard curve using a positive control sample." On page 15 there is a supposed to be a figure of a standard curve but I don't see one. My questions are: 1) is what do you use as a positive control? Would I use MCF7 cells treated with rotenone? 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.

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Answer

Thank you for contacting us at Abcam Scientific Support. I am happy to assist you.



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.

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Question

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

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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

can i purchase cytochrome c sepatately?

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Answer

Thank you for contacting us.

Cytochrome c is available in our catalog, ab140219

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Question

Hello,

I'm currently in the process of writing a paper that uses a protein quantification dipstick assay. We've developed our own dipstick with our antibodies that were not produced by Abcam/MitoSciences. I keep coming back to your image of a protein quantification dipstick (http://www.mitosciences.com/dipstick_protein_quantity_assays.html). Is there any way we could get permission to use this figure in a publication?

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Answer

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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Question

Hello

We recently order two kits to measure enzymatic activities (https://www.abcam.com/Complex-I-Enzyme-Activity-Dipstick-Assay-Kit-ab109720.html and https://www.abcam.com/Pyruvate-dehydrogenase-PDH-Enzyme-Activity-Dipstick-Assay-Kit-ab109882.html ).

theses kits allow us to determine enzymatic activites on a dipstick where the enzyme are previously immunocaptured form outr cells extracts.

We would like to know what subunits are targeted by theses kits. because we want to distinguish between activity and amount of enzyme. We are comparing wild type and mutant cell lines where some enzyme subunit are downregulated. we want to be sure that the complexex are not captured by these proteines.

thank you very much

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Answer

Thank you for contacting us.

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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Question

Inquiry: This is regarding the Complex I dipstick ELISA kit. I am planning to use this kit for assessment of Complex I activity in PBMCs extracted from human whole blood. Given the need for relatively high-throughput in assay performance, I was wondering if the Complex I activity assay was compatible with lysis detergents such as Triton, or if I would need to perform mechanical homogenization for these samples. Thank you for your time.

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Answer

Thank you for contacting us.
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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Question

I have been trying the Complex I Activity Dipstick Assay kit (ab109720), and although I follow the protocol carefully I have experienced some problems with the accuracy of the kit.

I run several samples at the same time (up to 16), and the major problem is that the samples (extract diluted in buffer A + buffer B) do not wick up into the stick with equal speed. Some samples are done within 15 minutes, while others can take up to 50 minutes. The concentration and the buffers are the same. I ran some of my samples in duplicates, and for the duplicates that had the same wicking speed, the results were good. For those where one of the parallells were done within 15 minutes and the other 30 minutes later, the signals would differ more, in spite of equal incubation in Activity buffer. Apart from the fact that this makes it very stressful when you run several samples (making sure none of the dipsticks run dry at any point), I do not trust the results when two parallells give such different results. I have made a standard curve to know that the assay is not saturated.

Do you have any tips concerning this problem? Can I add more buffer to the samples that have the highest wicking speed, or would this "dilute" the signal?

Any hints or tips would be appreciated!


Best regards,

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Answer

Thank you for contacting us.

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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Question

Hi, I'm looking to assay three cell types for mitochondrial protein quantity and activity. I would like to use the dipstick assays by MitoSciences (ab109720, ab109876, ab109722, and ab109877) but really do not need 30 tests of each--only 3. Is there an "assorted" pack I could purchase that just has one (or 5, or 10) of each of the four assays that are offered?

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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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