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

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:
  • Chavan H  et al. Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response. Oxid Med Cell Longev 2017:9251303 (2017). Read more (PubMed: 28163822) »
  • Barquissau V  et al. Reactive oxygen species enhance mitochondrial function, insulin sensitivity and glucose uptake in skeletal muscle of senescence accelerated prone mice SAMP8. Free Radic Biol Med 113:267-279 (2017). Read more (PubMed: 29024807) »

See all 26 Publications for this product

Customer reviews and Q&As

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...

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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 ...

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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.
Username

Mr. Christian Marx

Verified customer

Submitted Nov 15 2013

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 who...

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Thank you for contacting us.

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 y...

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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 nitr...

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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 subun...

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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 ...

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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 ...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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