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

31-40 of 86 Abreviews or Q&A

Answer

Thank you for contacting us.

You may use whole cell lysates which have been homogenized with Dounce homogenizer in PBS with addition of a protease inhibitor cocktail . For best result, we recommend that the assay is performed on the same day.


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 your enquiry which has been forwarded to me as my colleague, who you spoke to on the phone, is away from the office today.

We would usually recommend to purchase RHM homogenate (ab110347) as positive control sample. I can recommend to review the online datasheet for further information:

https://www.abcam.com/index.html?datasheet=110347 (or use the following: https://www.abcam.com/index.html?datasheet=110347).

The purified complex I or IV proteins are not provided in the kits because we are not able to find a good source for those proteins.

If you have tested the purified complex I and IV proteins with these kits before and proved they work, by all means you can use them as a control.

I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Question
Answer

Thank you for contacting us.

Samples may be prepared as purified mitochondria, crudemitochondria. In most cases whole tissue or cell lysates aresuitable but these may require some sample optimization. Mitochondria samples can be prepared bysimple differential centrifugation of homogenized samples. While any crude isolation may be effective, we recommend ourhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html(with Dounce homogenizer) if using cultured myocytes such as HL-1 cells.If using heart tissues we would recommendhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html(with Dounce homogenizer).



This kit was optimized to extract complex I into its native form (with all 47 subunits assembled in to a large complex). The sample extraction is native so care needs to be taken to extract the samples at equivalent starting concentrations to maintain the protein: detergent ratio and maintain enzyme integrity and activity.Specifically for this product - the extraction is performed by adding 1/10 volume of the 10X Detergent to samples at 5.5mg/mL



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.

Same process for converting all to molarity per amount of protein loaded into well.
Note these are all per cm and should be converted into path length for microplate which should be about 0.6cm.

Extinction coefficients

ab109908 (complex II enzyme activity),
ε600 = 21 mM-1cm-1

ab109909 (complex IV activity),
ε550 = 21.1 mM-1cm-1

ab109716 (ATP synthase activity)
ε340 = 6.220 mM-1cm-1

ab119692 (citrate synthase activity).
Ε412 = 14.1 mM-1cm-1

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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https://www.abcam.com/abreviews

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Question
Answer

Thank you for contacting us.


My colleague in lab has replied as follows:

If a user does not want to compare relaive mOD/min/amount loaded into each well – another unit can be calculated: The enzyme in question is bound to the well oxidizes NADH while simultaneously reducing the dye in the buffer in a 1:1 ratio.

This leads to an increase in absorbance at 450 nm (dye Epsilon 450nm = 37 mM-1 cm-1).

The height of 200 uL of liquid in each well is approximately 0.6 cm, therefore epsilon is 22 mM-1 well-1.

So the change in abs per minute in each well divided by 22 is the mM oxidized per minute. This can be divided by the amount of material loaded into the well in microg to get units that all samples can be compared using,

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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https://www.abcam.com/abreviews

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Question
Answer

Thank you very much for your interest in our products.

I just sent you Proforma Invoice (quotation) #45550 for the 4 kits. This quote will be good until Sept 30th when the discount expires. When you're ready to place the order, just call us and mention this number. If you need more reagents for your studies, you can use the discount code 4FOR3-TB3ME through Sept 30th to get the 4 for the price of 3 promotion on all products.

We would be very grateful if you could submit an Abreview aboutthese productsvia the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers. You would have to do this manually, by working with our Scientific Support team through mailto:us.technical@abcam.com.

To find out more about our Abreview system, please see the following webpage: https://www.abcam.com/abreviews

And Abpoints: https://www.abcam.com/index.html?pageconfig=loyalty_rewards_list

We hope to receive your Abreviews. Thank you for your time and effort.

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Answer

Thank you again for keeping me updated about your situation.

I've checked with the lab, and they use water purified by a Barnstead E-Pure Model D4641 purification system.As long as you're using purified water (preferably Molecular Biology or Tissue Culture grade),we don’t think that the water should affect the background signal.

What were theCV or standarddeviationvalues for the blank values that you found with the old and new kits? These values do seem in the range of normal background levels. Please keep me updated with any results with the kit, and let me know if there is anything else that we can do for you.

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Question

Respected Madam, I am sorry for not contacting you earlier. But I did
only one exp and was going to repeat another. I did not use mouse
heart mito but I used mito extracted from MCF-7 culture cells. So I
was waiting for an exp with mouse heart mito before I contact with you
again. We did mito from MCF-7 culture cells, T-cells, mouse heart and
tumor. They all require different amounts of mito and different
incubation times for color developing. Heart develops color
immediately but MCF-7 cells take upto 2 hrs to develop color. (MCF-7
cells have very little mito) That's why I was worried about my blank.
So here I am writing down (raw data) the abs of my blank over time.
Old kit: blank - 0.083 (after 1hr), 0.101 (after 2hr), 0.110 (after 3hr)
New Kit: blank - 0.102 (after 30min), 0.130 (after 1hr), 0.189 (after
2hr) (Have not used the kit again after one exp)
The new kit blank seems to be developing color faster. Is there a pH
that I am to be controlling? I will be doing another exp in the next 2
weeks and then will send data again. I also have a question. Should we
be using deionized water or autoclaved water for reconstitution of any
of the buffers in the kit? Does it make a difference? If so how?
I cannot use a lot of mito to make my sample reaction go faster
because then I am unable to see the subtle differences between the
different conditions/samples. I hope I was able to state my case
clearly. I will be in touch. Thank you so much for the support. Have a
nice day. bye

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Answer

Thank you very much for your reply and for keeping me updated.

Thank you also for sending the additional information about your experiment; I think I better understand your situation now. I have asked the lab for more details about the water that they used to prepare the solutions in the previous kits. As far as I know, this was neutral, deionized water. You don't need to be controlling the pH during the reaction.

Please keep me updated with any new results, and I will be back in touch once I hear from the lab. If there is anything else that we can do for you, please let me know and I'll be happy to help.

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Answer

Thank you for contacting us.

The lab let me know that +4C at either speed would befine for both kits.

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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Answer

These solutions are not the same… different buffers and 10-fold different detergent concentrations.

I hope this is helpful. Please contact us again if you have any further questions.

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31-40 of 86 Abreviews or Q&A

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