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

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.

Images

  • 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.

  • 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.

  • 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.
  • 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.

Protocols

References

This product has been referenced in:
  • Varela-Lopez A  et al. Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive, sunflower or fish oils. J Nutr Biochem 52:36-44 (2018). WB ; Rat . Read more (PubMed: 29144994) »
  • Arias-Mayenco I  et al. Acute O2 Sensing: Role of Coenzyme QH2/Q Ratio and Mitochondrial ROS Compartmentalization. Cell Metab 28:145-158.e4 (2018). Read more (PubMed: 29887397) »
See all 32 Publications for this product

Customer reviews and Q&As

1-10 of 22 Abreviews or Q&A

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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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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Complex I activity assay in HCT116 cells

Good Good 4/5 (Ease of Use)
Abreviews
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.

Mr. Christian Marx

Verified customer

Submitted Nov 15 2013

Question
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
Answer

Thank you for contacting us.

Cytochrome c is available in our catalog, ab140219

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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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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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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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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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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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