Complex II Enzyme Activity Microplate Assay Kit (ab109908)

Reviews (2)Q&A (51)References (65)
- Complex II Enzyme Activity Microplate Assay Kit (ab109908)
  • - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
  • Functional Studies - Complex II Enzyme Activity Microplate Assay Kit (ab109908)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 3 hr
  • Sample type: Cell Lysate, Purified mitochondria, Tissue Lysate

Overview

  • Product name

    Complex II Enzyme Activity Microplate Assay Kit
    See all Complex II kits

  • Detection method

    Colorimetric

  • Sample type

    Cell Lysate, Tissue Lysate, Purified mitochondria

  • Assay type

    Enzyme activity

  • Assay time

    3h 00m

  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human

  • Product overview

    Complex II Enzyme Activity Microplate Assay Kit is designed for determining the Complex II activity in a human, mouse, rat or bovine sample. Each of the 96 wells in the kit has been coated with an anti-Complex II monoclonal antibody (mAb) which purifies the enzyme from a complex sample such as mitochondria, tissue homogenate or cell lysate. After this in-well purification the production of ubiquinol by the enzyme is coupled to the reduction of the dye DCPIP (2,6-diclorophenolindophenol) and a decreases in its absorbance at 600 nm, which in turn recycles the substrate ubiquinone.

  • Notes

    Succinate, Ubiquinone 2, DCPIP and Lipid?Phospholipd Mix should be stored at -80°C. All other components should be stored 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

  • - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
    - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
    Figure 2. This assay is compatible with different sample types such as mitochondria, tissue or cell lysates and in multiple species including human and rodent samples. Typical linear range data are shown for ab109908.
  • - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
    - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
    Figure 1. Example of raw data. Note the lag period before activity. Also note the activity of mitochondria (BHM, bovine heart mitochondria) is higher than whole cell lysate (HepG2, human hepatoblastoma) and the reaction ends at >1600 seconds because the substrates are used up.
  • Functional Studies - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
    Functional Studies - Complex II Enzyme Activity Microplate Assay Kit (ab109908)
    Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. 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.

    Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, substrate is added, and enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed). 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.

References (65)

Publishing research using ab109908? Please let us know so that we can cite the reference in this datasheet.

ab109908 has been referenced in 65 publications.

  • Black M  et al. FOXM1 nuclear transcription factor translocates into mitochondria and inhibits oxidative phosphorylation. Mol Biol Cell N/A:mbcE19070413 (2020). PubMed: 32348194
  • Wang J  et al. Fundc1-dependent mitophagy is obligatory to ischemic preconditioning-conferred renoprotection in ischemic AKI via suppression of Drp1-mediated mitochondrial fission. Redox Biol 30:101415 (2020). PubMed: 31901590
  • Wang J  et al. Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2. Theranostics 10:384-397 (2020). PubMed: 31903127
  • Agarwal E  et al. Myc-mediated transcriptional regulation of the mitochondrial chaperone TRAP1 controls primary and metastatic tumor growth. J Biol Chem 294:10407-10414 (2019). PubMed: 31097545
  • Li J  et al. Melatonin attenuates renal fibrosis in diabetic mice by activating the AMPK/PGC1a signaling pathway and rescuing mitochondrial function. Mol Med Rep 19:1318-1330 (2019). PubMed: 30535482
View all Publications for this product

Customer reviews and Q&As

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1-10 of 53 Abreviews or Q&A

Question

On page 8 steps 3 and 4, the protein concentration needs to be determined before the detergent is added. How do you ensure the samples are at 5.5 mg/mL?

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Answer

Thank you very much for your call today and for your patience while I have been in touch with the lab scientists regarding this enquiry.

If you're using a tissue homogenate or whole mitochondria sample, before extraction with the kit’s detergent (lauryl maltoside)the protein concentration of the sampleshould be 5.5mg/mL.A way of doing this is as follows:

Sacrifice a small portion of the sample (10 – 20uL), solublize it with any ionic detergent of choice and then determine protein concentration in this small aliquot. That number will be representative (taking into account any potential dilutions made) of the protein concentration of the original sample.

If the equivalent concentration was higher than 5.5mg/mL add more buffer accordingly. If the equivalent concentration was lower than 5.5mg/mL centrifuge the whole tissue homogenate or whole mitochondria at high speed. This will generate a pellet which can then be resuspended in a smaller volume.

Once the sample is at the right concentration,you can add the kit’s detergent and follow the instructions



We have a booklet that explains in more detail sample extraction. This booklet can be found in the following link:

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

I hope that this information will be useful, but if please let me know if you have any further questions or need anything else and I'll be happy to help.

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Question

I'm using this kit, but I haven't determined the protein concentration. I'm trying to find the right cell number to use. I'm using 100,000, 1 million, and 10 million lymphoma cells. I tried something similar before, but the assay didn't work (no signal above negative control). Do I have to dilute my sample in the incubation buffer in order for the assay to work? I want to make sure it's concentrated enough, and I haven't measured it to the recommended 1.2 mg/ml, so is there some sort of ratio I could follow (i.e. 50 ul sample, 10 ul incubation buffer)?

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Answer

Thank you for your inquiry.


The assays can be optimized by cell number, but since all cells are different sizes we use total protein mass.


In general large cells such as fibroblast, HeLa etc are about 0.25 mg/million. So for a normal cell, 10 million would work.

However lymphoblasts are almost always very small cells and would require even more cells.

One solution is to resuspend the cell pellet to 10 volumes with extraction buffer to get a protein concentration of 2-5 mg/mL.

So to get a 200 uL sample you need to start with 20 uL cell pellet + 180 uL lysis buffer.

We would always recommend using a positive control – a human cell line e.g. HeLa or one of our mitochondrial samples (below)

Name
Species
Applications
Amounts
MitoSciences code
Abcam code

https://www.abcam.com/ab110351
Ms
Immunocapture, BNPage
2 mg
MS822
https://www.abcam.com/ab110351

https://www.abcam.com/ab110348
Rat
Immunocapture, BNPage
2 mg
MS819
https://www.abcam.com/ab110348

https://www.abcam.com/ab110338
Cow
Immunocapture, BNPage
2 mg
MS802
https://www.abcam.com/ab110338

https://www.abcam.com/ab110350
Ms
Immunocapture, BNPage
2 mg
MS821
https://www.abcam.com/ab110350

https://www.abcam.com/ab110347
Rat
Immunocapture, BNPage
2 mg
MS818
https://www.abcam.com/ab110347

https://www.abcam.com/ab110349
Ms
Immunocapture, BNPage
2 mg
MS820
https://www.abcam.com/ab110349

https://www.abcam.com/ab110346
Rat
Immunocapture, BNPage
2 mg
MS811
https://www.abcam.com/ab110346


I hope this information helps. Please contact us with any other questions.

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

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Abreviews
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Easy to follow instruction and standardize using cell lysate. Good sensitivity with microplate reader

Abcam user community

Verified customer

Submitted Feb 27 2018

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

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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.

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Question

Hi,
 
With regards to using Malonate (Buffered Malonic Acid) for an inhibitor of Complex II what concentration and pH is recommended? Thanks.
 

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Answer

According to the literature, malonate inhibits complex II with an IC50 of ˜40 μM (PMCID: PMC2507763). Please let me know if you have any further questions!

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Question



Inquiry: Hello, I recently purchased this kit, and I have a few questions before proceeding with my pilot experiment. 1.) Should I keep samples (cells) on ice while I am resuspending them? 2.) Should I add a protease inhibitor to the detergent? 3.) I have isolated mito for a positive control (to ensure the kit works). However, I am trying to come up with a positive control for increased activity of Complex II. Do you know of any compounds or even amino acid mutations that allow for increased activity of Complex II? 4.) I would like to use a negative control- that decreases activity of Complex II. In the protocol, you mention TTFA as an inhibitor of Complex II. Can I add the TTFA to the activity solution and decrease the amount of activity buffer used? 5.) Do you know if post-translational modifications like acetylation are maintained on Complex II bound to the mAb? Thanks for all your help!

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Answer

Thank you for contacting us.

Yes, you should keep samples on ice, and also add a protease inhibitor to the detergent.

Yes TTFA can be added and should decrease the activity of Complex II.
However TTFA is not as specific/effective as some of the other OXPHOS inhibitors. Malonate (Buffered malonic acid) could also be used.
Acetylation should be preserved. The capture antibody is a mouse antibody and we have been able to see acetylation by adding a rabbit anti-acetyl lysine antibody ab21623 and Goat anti-rabbit HRP to the plate.
https://www.abcam.com/acetyl-Lysine-antibody-ChIP-Grade-ab21623.html


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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Question

Phone call: How to analyse the concentration of cell lysates?

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Answer

Pipette up and down cells several time for complete lysis and then determine the protein concentration using BCA or Bradford method. Once protein concentration is known then please follow rest of the procedure.

20 million of average sized cells per ml will give 5mg/ml of protein.

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Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs.

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
30ug Lung mitochondrion samples from mice in buffer containing 250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.25% protease inhibitor, and 0.5% BSA, pH 7.4 were used for the assay.

Dr. Amit Agarwal

Verified customer

Submitted Jul 24 2013

Question

Customer is trying to calculate rate of activity in mM/min/mg protein as seen in fig.11 of  paper Li C  et al. Free Radic Biol Med 60C:29-40 (2013). (PubMed: 23376468, http://www.sciencedirect.com/science/article/pii/S0891584913000233) In order to do this they are asking if we can provide the concnetration of DCIP provided in this kit.

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Answer

The DCIP concentration is 2.5mM.

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Question

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

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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.

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