Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)

Overview

  • Product name
    Complex I Enzyme Activity Microplate Assay Kit (Colorimetric)
    See all Complex I kits
  • Detection method
    Colorimetric
  • Sample type
    Cell culture extracts, Tissue
  • Assay type
    Enzyme activity
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    Complex I Enzyme Activity Assay Kit (ab109721) is a kit designed for the analysis of mitochondrial OXPHOS Complex I enzyme activity from human, rat, mouse and bovine cell and tissue extracts.
    Capture antibodies specific for Complex I are pre-coated in the microplate wells. Samples are added to the microplate wells which have been precoated with a specific capture antibody. After the target has been immobilized in the well, Complex I activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.


    Complex I activity is controlled by enzyme amount and by post-translational phosphorylation at key specific regulatory residues. Cellular metabolism governs these two factors. Ultimately, the cell type and growth conditions will affect Complex I activity measurements.


    Note: This activity assay measures the diaphorase-type activity of Complex I. This activity is not dependant on the presence of ubiquinone and therefore inhibitors, such as rotenone, which bind at or near the ubiquinone binding site do not inhibit this assay. However, both the activity assay and the quantity assay described here are affected by enzyme assembly deficiencies.


    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Notes

    ab109721 is shipped at 4°C. 20X NADH and 100X Dye are shipped lyophilized. Rehydrate 20X NADH by adding 1.1 mL H2O. Rehydrate 100X Dye by adding 0.25 mL H2O, then vortex each thoroughly until dissolved. After hydration unused amounts of these two materials should be stored at -80°C. When planning multiple experiments, aliquot these reagents to prevent freeze thaw cycles. Store all other components at 4°C

    Related products

    Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Platform
    Microplate reader

Properties

Images

  • ab109721 measures Complex I activity in human cultured cells within the recommended ranges given in the protocol. Example of Complex I activity measured in Hep2 cells is showed. (Note that these ranges depend on mitochondria preparation quality).

  • ab109721 measures Complex I activity in human and rat cultured cells but also in tissues/tissue mitochondria samples within the recommended ranges given in the protocol. Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).

  • Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).

  • (A) ab109721  was used to measure Complex I activity in normal and Rho0 human fibroblast whole cell lysates at 1mg/mL. Rho0 cells are cells in which the mitochondrial DNA has been removed and therefore essential Complex I proteins are not expressed. As shown, the rho0 cells showed no/little complex I activity.

    (B) In a similar analysis, rat cardiomyocytes were grown for 5 days in ± 40 µM chloramphenicol (CAM) to inhibit mitochondrial protein synthesis, Complex I assembly and hence activity was greatly reduced in samples loaded at 0.5 mg/mL whole cell lysates.

  • Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Protocols

References

This product has been referenced in:
  • Zhang XL  et al. RNAi-mediated knockdown of DJ-1 leads to mitochondrial dysfunction via Akt/GSK-3ß and JNK signaling pathways in dopaminergic neuron-like cells. Brain Res Bull N/A:N/A (2019). Read more (PubMed: 30634017) »
  • Xu D  et al. Novel role of mitochondrial GTPases 1 in pathological cardiac hypertrophy. J Mol Cell Cardiol 128:105-116 (2019). Read more (PubMed: 30707992) »
See all 72 Publications for this product

Customer reviews and Q&As

41-50 of 86 Abreviews or Q&A

Answer

Thank you for your phone call. Preparing the samples at 12,000 g will be suitable for both kits. I hope this helps, please let me know if you need any additional information or assistance.

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Answer

To determine the amount of NADH oxidized by PDH – we need to convert the color change of the dye to NADH oxidized.
These react in a 1:1 ratio. The molar extinction for the dye is 25.9/ mM / well


So divide the increase in absorbance in each well at 450 nm by 25.9 to determine mM NADH oxidized in that period of time.
I hope this is helpful. Please contact us again if you have any further questions.

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Answer

Vielen Dank für Ihre Anfrage.

Sie haben Recht, ist der Wert OD pro Minute höher in Probe 1 als in Probe 2 zeigt der Komplex I eine höhere Aktivität in Probe 1 als in Probe 2.

Die Aktivität wird folgendermaßen gemessen:

Das Enzym (Komplex I) oxidiert NADH zu NAD+. Dies führt zu einer simultanen Reduktion eines Farbstoffes, was wiederumeiner erhöhtenAbsorption (OD) bei 450nm führt.

Kurz: hohe OD, hohe Enzymaktivität. :-)

Ich hoffe, dies beantwortet Ihre Frage und ich wünsche Ihnen viel Erfolg mit Ihren Experimenten.

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Answer

Vielen Dank für Ihre Anfrage.

ab109721 ist ein Kit, dass die Aktivität von Complex I Enzym messen kann.

Leider ist uns und dem Labor kein spezifisches Stimulans für Complex I bekannt. Er sollte aktiv sein in einer normalen gesunden Zelle.

Da in Ihren Experimenten die Hemmung funktioniert, nehme ich an, dass die ungehemmten Proben dann eine positive Aktivität zeigen.

Dieses Kit ist mit folgenden Proben getestet worden: Mitochondrien aus Rattenherz, Leber und Hirn, Maus Hirn und Leber, humane Fibroblasten und HepG2 Zellen. (siehe Protokoll)

Die Austestung verschiedener Behandlungen und Zellkulturbedingungen können wir leider nicht leisten, da es sich ja hierbei um Forschungsfragen handelt.

Es tut mir leid, dass ich keine ausgiebigeren Informationen für Sie habe und hoffe, dass diese dennoch hilfreich sind.

Ich wünsche Ihnen viel Erfolg beim Austesten der optimalen Bedingungen für Ihre Versuche und verbleibe mit

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Answer

Thank you again for sending your data. I have heardagain from the MitoSciences lab and I wanted to follow-up with you.

According to the QC report for the lot that you previously tried, the components do appear yellow after lyophilization, but the background signal was low compared to the sample readings.They also measured this assay kinetically in mOD/min.Could you tell me how long the assay was run to obtain the OD values that you sent? What were the sample readings?

If the problem persists with the replacement kit, I can send some positive control bovine heart mitochondrial lysate, which is what the lab uses to test these kits. Please keep me updated about the results with this kit, and let me know if you need anything else from me.

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Answer

Thank you very much for your reply and for sending the file again.

Do you have data for the entire kit that was run, including sample readings? It will be helpful to see the difference between the blank and the sample readings.

Also, the MitoSciences lab would like to know if the reagents were used immediately after reconstitution, or were they stored? If they were stored, could you let me know the storage temperature and length of time they were stored?

Have you had a chance to move forward with the replacement kit? I'm wondering if the reconstitued reagents are yellow again. The lab had intended to re-test the lot that you previously tried, but we have sold out of this lot already. We haven't had reports of similar problems, so we are hoping it was an isolated incident. Please keep me updated about the new kit.

I look forward to hearing from you. If there is anything else you need, please don't hesitate to ask. Have a nice day!

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Answer

Thank you very much for your reply.

I could not find the XLS file- can you check that it was attached to your previous email? The MitoSciences lab has decided to re-test a kit from the lot that gave high background in order to check that it is functioning correctly. I will have an update from them as soon as possible.

Thank you again for letting us know about the trouble with this kit, and please let me know if there is anything else that we can do for you.

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Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab89782.

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.

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Answer

Thank you very much for your call today and for letting us know about the trouble with this kit.

I have contacted the MitoSciences lab for suggestions to improve the results, and if you have any data it would be very helpful. As we discussed, I'm also sending a free of charge kit on the order *** which should arrive tomorrow.

Please let me know if you have further questions or if there is anything else that we can do for you, and I'll be happy to help.

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Answer

Thank you for contacting us.

We have several assay kits directed towards detecting the Complex IV activity. These can be accessed from the following links:

https://www.abcam.com/Complex-IV-Human-Specific-Activity-Microplate-Assay-Kit-ab109910.htmlhttps://www.abcam.com/Complex-IV-Human-Specific-Activity-Microplate-Assay-Kit-ab109910.html https://www.abcam.com/Complex-IV-Human-Specific-Activity-Microplate-Assay-Kit-ab109910.html

https://www.abcam.com/Complex-IV-Rodent-Enzyme-Activity-Microplate-Assay-Kit-ab109911.htmlhttps://www.abcam.com/Complex-IV-Rodent-Enzyme-Activity-Microplate-Assay-Kit-ab109911.html https://www.abcam.com/Complex-IV-Rodent-Enzyme-Activity-Microplate-Assay-Kit-ab109911.html

https://www.abcam.com/Complex-IV-Human-Enzyme-Activity-Microplate-Assay-Kit-ab109909.htmlhttps://www.abcam.com/Complex-IV-Human-Enzyme-Activity-Microplate-Assay-Kit-ab109909.html https://www.abcam.com/Complex-IV-Human-Enzyme-Activity-Microplate-Assay-Kit-ab109909.html

As well as immunocapture kits:

https://www.abcam.com/Complex-IV-Immunocapture-Kit-ab109801.htmlhttps://www.abcam.com/Complex-IV-Immunocapture-Kit-ab109801.html https://www.abcam.com/Complex-IV-Immunocapture-Kit-ab109801.html

https://www.abcam.com/Complex-IV-Rodent-Immunocapture-Kit-ab109860.htmlhttps://www.abcam.com/Complex-IV-Rodent-Immunocapture-Kit-ab109860.html https://www.abcam.com/Complex-IV-Rodent-Immunocapture-Kit-ab109860.html

When carrying out such assays (including that of ab109721)it is recommended that all reading be performed in duplicate. This ensures more accurate readings and highlights and anomalous results obtained through errors in carrying out the assay.

I hope this information has been of help. If you have any further questions, please do not hesitate to ask.

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41-50 of 86 Abreviews or Q&A

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

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