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 102 Publications for this product

Customer reviews and Q&As

21-30 of 86 Abreviews or Q&A

Answer


To convert from mOD/min to nmol/min you can use the extinction coefficient for the dye in that kit.
For the complex I kit (ab109721), the dye Epsilon at 450nm = 37 mM-1 cm-1

The height of 200 uL of liquid in each well is approximately 0.6 cm, therefore epsilon is 22.2 mM-1 well-1 for the complex I kit.

For the complex II kit (ab109908), the dye Epsilon at 450nm = 21 mM-1 cm-1, which for 200uL of liquid (0.6 cm) equates to 12.6 mM-1 well-1.

So the change in absorbance per minute in each well divided by 22.2 for complex I or 12.6 for complex II is the mM oxidized per minute.

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Answer

It is possible that NADH could be monitored (decrease at 340 nm); however, the reaction may need an acceptor molecule to receive the reductant. In this kit tetrazoleum salt (WST1) is used as a suitable acceptor dye and this kit is a relatively sensitive method for use with many samples from different origins. The oxidation of NADH by complex I dehydrogenase leads to the reduction of tetrazoleum salt to a more colored formazan dye.

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Answer

Thank you for contacting us and your interest in our products. I am sorry for the delay in getting back to you.

The three kits mentioned (ab109721, ab109911 and ab109908) use 10% lauryl maltoside detergent to prepare the sample at 5 mg/mL. Therefore this is interchangeable between all three kits.

In regards to your second question, we find simply adding the buffer to the lipid mixture is enough. There should not be a need for mixing.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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Question
Answer

We have never tested this kit with Triton-X 100 solubilized samples, however it would very likely prevent activity. I would suggest starting with a sample that does not contain any detergent. The detergent we provide with the kit is optimal for solubilization of the whole complex in its native form, which is critical for activity.

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Answer

Thank you for contacting us.

We have been in contact with the lab regarding preparation of lysates. It appears that Table 1 is earlier in the text on page 5 and Sample preparation is in section 6. I have pasted furhter information below.

With a cell pellet or tissue homogenate in PBS:

6.3 Determine the sample protein concentration using a

standard method such as BCA method. Adjust the

concentration of the sample to 5.5 mg/ml using PBS. For

details of sample solubilization conditions see frequently

asked questions section.



6.4 Extract the proteins by adding Detergent. To do this add

1/10 of a volume of Detergent (e.g. if the total sample

volume is 500 μl, add 50 μl of Detergent). Mix well. Place

the tube on ice for 30 minutes to allow solubilization. Note

the sample concentration is now 5 mg/ml.





I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Thank you for your enquiry.


I can confirm that In each of these kits we use a non-ionic native detergent at an optimized concentration to maintain OXPHOS complex assembly and activity. This detergent is 1% final N-dodecyl-b-D-maltoside (aka lauryl maltoside). This detergent has been used for many years, particularly in the field of mitochondrial biology and in blue native PAGE separation of OXPHOS complexes.

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

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Question
Answer

Thank you for contacting Abcam.

The capture antibody in the Complex I Enzyme Activity Microplate Assay Kit (ab109721) binds complex I in a native state with all subunits present as tested by mass spectroscopy.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Hello!

The 10X detergent is not diluted before adding to samples at 1/10 volume.

More samples can be prepared by scaling the volumes down. A total of 10mL of sample can be prepared

So 10 samples at 1mL, 20 samples at 0.5 mL, 40 samples at 0.25 mL etc. I wouldn’t recommend preparing too many samples since replicates are encouraged.

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Answer

Thank you for your inquiry.
The 10xdetergent of this kit is Lauryl maltoside 10%.
Thank you.

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Answer

Thank you for contacting Abcam.

Unfortunately we do not have any information about ab109721 ab109721 working with mouse striatal tissue punches. We know it will work with mouse tissue, but we do not know if there will be a high enough concentration of mitochondrial proteins in your sample of interest to get a signal.

If possible, it may be a good idea to pool as many mouse striatal tissue punches together as possible and then use that as your sample in the assay.


I am sorry that I am not able to provide more specifics on this question, but if there is anything else I can help you with, please let me know.

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21-30 of 86 Abreviews or Q&A

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