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    products/assay-kits/complex-i-enzyme-activity-microplate-assay-kit-colorimetric-ab109721.pdf

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

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (4)Q&A (82)References (219)

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Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
  • Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.
  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 3 hr 30 min
  • Sample type: Cell culture extracts, Tissue

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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 dependent 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.


    Chinese protocol available. See protocols section below.

  • 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

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 96 tests
    100X Dye (lyophilized) 1 unit
    10X Blocking Solution 1 x 10ml
    Detergent 1 x 1ml
    20X Wash Buffer 1 x 25ml
    20X NADH (lyophilized) 1 unit
    96-well microplate (12 strips) 1 unit
  • Research areas

    • Signal Transduction
    • Metabolism
    • Mitochondrial
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Other Metabolism Assay
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Mitochondrial markers
  • Alternative names

    • NADH dehydrogenase

Associated products

  • Positive Controls

    • Bovine Heart Mitochondria (ab110338)
    • Rat liver tissue lysate - mitochondrial extract (ab110346)
    • Rat heart tissue lysate - mitochondrial extract (ab110347)
    • Rat brain tissue lysate - mitochondrial extract (ab110348)
    • Mouse liver tissue lysate - mitochondrial extract (ab110349)
    • Mouse heart tissue lysate - mitochondrial extract (ab110350)
    • Mouse brain tissue lysate - mitochondrial extract (ab110351)

Images

  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)

    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).

  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)

    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).

  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)

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

  • Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.
    Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.Image courtesy of Yao X et al.PLoS One. 2015; 10(10): e0139416. doi: 10.1371/journal.pone.0139416.

    Mitchondiral fractions from the heart tissue of Rats infected by S. pneumoniae, or given PBS sham control, were subjected to measurements of complex I-V activities. Complex I was measured with ab109721 (top left), Complex II + III were measured using ab109905 (top right), Complex IV was measured using ab109911 (bottom left) and Complex V was measured using ab109714 (bottom right).

    Freshly isolated mitochondrial pellets were resuspened in PBS supplemented with 10% detergent provided in the kits. Protein concentrations of these mitochondrial lysates were estimated and 25 μg (for complex I, IV and V) or 100 μg (for complex II+III) mitochondrial protein was used per reaction. Enzyme activities were measured spectrophotometricly in triplicate and expressed as changes of absorbance per minute per mg protein.

  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)

    (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.

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

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (219)

Publishing research using ab109721? Please let us know so that we can cite the reference in this datasheet.

ab109721 has been referenced in 219 publications.

  • Zhu Y  et al. miR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1. Oxid Med Cell Longev 2022:3182931 (2022). PubMed: 35126811
  • Lei M  et al. Long non-coding RNA TUG1 sponges microRNA-9 to protect podocytes from high glucose-induced apoptosis and mitochondrial dysfunction via SIRT1 upregulation. Exp Ther Med 23:236 (2022). PubMed: 35222713
  • Kim M  et al. Evaluation of Parkin in the Regulation of Myocardial Mitochondria-Associated Membranes and Cardiomyopathy During Endotoxemia. Front Cell Dev Biol 10:796061 (2022). PubMed: 35265609
  • McElroy GS  et al. Reduced expression of mitochondrial complex I subunit Ndufs2 does not impact healthspan in mice. Sci Rep 12:5196 (2022). PubMed: 35338200
  • Franchina DG  et al. Glutathione-dependent redox balance characterizes the distinct metabolic properties of follicular and marginal zone B cells. Nat Commun 13:1789 (2022). PubMed: 35379825
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 86 Abreviews or Q&A

Complex I activity in HCT116 cells

Excellent Good 4/5 (Ease of Use)
Abreviews
Abreviews
abreview image
HCT116 cells were cultivated in standard DMEM medium for 48 hrs. Cell preparation was performed as described in the protocol. Protein concentration in each sample was quantified and adjusted by Bradford assay.
We looked at complex I activity dependent on the protein amount applied to the plate (fig.) thus we would suggest to use protein amounts around 200 µg for this assay.
The kit is easy to use but takes a long time to perform. The results seem to be reliable.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Mr. Christian Marx

Verified customer

Submitted Dec 12 2013

control of mitochondrial function and cell growth by the atypical cadherin Fat1

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Very easy to follow instructions and to standardize using cell lysates. Good specificity with microplane reader.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Feb 28 2018

Chronic hypoxia disrupts mitochondrial function in the guinea pig placenta

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
I used isolated protein from guinea pig placenta to perform the experiment with this kit. Complex I enzyme activity can be detected.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Feb 07 2017

Question

How the sample should be prepared?

Read More

Abcam community

Verified customer

Asked on Mar 26 2012

Answer

Thank you for contacting us.

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.

Step 3 – in this we recommend adjusting the lysed cell sample protein concentration to approximately 5.5 mg/mL as determined by some form of protein assay (you can take a small % of sample for this – it may need some prior lysis with SDS or other detergent based on the nature of the sample or protein assay conducted).

Step 4 -Once adjusted it is time for the protein extraction using our supplied extraction detergent.

Wewould recommend doing both steps – step 3 adjustment is precautionary – step 4 is essential to normalize different samples loaded into the plate wells,

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

Read More

Abcam Scientific Support

Answered on Mar 26 2012

Question

I am planning to investigate the OXPHOS pathway in rat heart tissue. I have looked at your complex assays (ab109721, ab109908, ab109911 and ab109716) and your mitochondrial isolation kit for tissue (ab110168) and had a few questions: 1. Are the mitochondria isolated from the kit suitable for use on all of the above assays? 2. Is there any further treatment that is required before the isolated mitochondria can be used in each assay (i.e. incubation with detergent to isolate protein complex) or are they used directly into the plate? Thanks

Read More

Abcam community

Verified customer

Asked on Nov 16 2015

Answer


1) Yes, the mitochondrial isoaltion kit is suitable for use with all kits listed.

2) Suspend the prepared mitochondria to 5.5 mg/mL as described in the attached document and follow from step 5 (adding detergent to mitochondia) to generate an extract suitable for all kits.

Read More

Heather Allen

Abcam Scientific Support

Answered on Nov 16 2015

Question

Is the RIPA buffer compatible with this kit?
What is the recommended lysis buffer?

Read More

Abcam community

Verified customer

Asked on Jul 19 2013

Answer

I would not recommend RIPA buffer because it contains SDS a powerful ionic detergent. The MS141 ab109721 Complex I activity assay contains a mild non-ionic detergent lauryl (n-dodecyl) maltoside which preserves Complex I activity.

Please check the sample preparation guide as well:

http://www.mitosciences.com/PDF/mitosciences-sample-preparation-guide.pdf

Read More

Padamjeet Singh

Abcam Scientific Support

Answered on Jul 19 2013

Question

Do you have any recommendations for OXPHOS assays made specifically for blood?

Read More

Abcam community

Verified customer

Asked on May 24 2013

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.

Read More

Abcam Scientific Support

Answered on May 24 2013

Question

I read the protocols of both kits (109721 and 109720). For preparing tissue samples, in dipstick kit, there are procedures about how to prepare, while in microplate kit, there is no relative procedure indicating how much tissue to start with and which buffer to use to homogenize the tissue.

Can you explain more of those questions?

Read More

Abcam community

Verified customer

Asked on Feb 02 2012

Answer

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

Read More

Abcam Scientific Support

Answered on Feb 02 2012

Question

I'd like to use the tissue, once processed using ab110169, for the following kits 1) ab109721, 2) ab110413 and ab109902.
So the main question is, can I store the brains by freezing (eg. liquid nitrogen to -80 deg C) and thaw the brains and produce extracts that will produce viable sample for use with all 3 kits noted above.

Read More

Abcam community

Verified customer

Asked on Aug 12 2014

Answer

Yes, you can extract mitos from frozen tissues but be aware that freezen/ thaw cycle would weaken the cell membrane and cause less recovery of intact mitochondria.

A: There will be slightly loss if you extract enzyme from frozen mitos versus fresh mitos due to the degradation. Adding protease inhibitor to your mito prep would minimize the loss.

As long as the tissues are properly frozen, the variant between different time points should be minimum (within weeks). However, multiple freezen/thaw cycle will cause difference. If you plan to extract sample from different days, make sure you make aliquots of your samples. if you plan to compare the enzyme activity from different samples, we highly recommend to prep the samples under the same condition.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Aug 12 2014

Question

Can the mitochondria isolated with ab110168 be frozen prior to the use with mitochondrial activity kits in general and with ab109721 in particular.

Read More

Abcam community

Verified customer

Asked on Oct 01 2013

Answer

I can confirm the the mitochondria prepared with ab110168 can be frozen and stored in -80 C to be compatible for subsequent analysis using ab109721 kit.

Read More

Abcam Scientific Support

Answered on Oct 01 2013

1-10 of 86 Abreviews or Q&A

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