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    products/assay-kits/complex-ii-enzyme-activity-microplate-assay-kit-ab109908.pdf

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Complex II Enzyme Activity Microplate Assay Kit (ab109908)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (2)Q&A (51)References (96)

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- 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

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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

  • Storage instructions

    Please refer to protocols.
  • Components 96 tests
    10X Blocking Solution 1 x 5ml
    Detergent 2 x 1ml
    20X Buffer 1 x 15ml
    Complex II Activity Buffer 1 x 25ml
    DCPIP/DCIP 1 x 250µl
    Phospholipids 1 x 6ml
    Pre-coated 96-well microplate (12 strips) 1 unit
    Succinate 1 x 500µl
    Ubiquinone 2 1 x 60µl
  • Research areas

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

    Complex II is also called succinate ubiquinone oxidoreductase or more commonly succinate dehydrogenase complex. This complex is composed of four nuclear encoded subunits and contains a flavin (FAD), non-heme iron centers and a b-type cytochrome as prosthetic groups. It is both a component of the electron transport chain and an enzyme of the Krebs cycle. Complex II deficiencies are seen in OXPHOS genetic disease and found in a type of cancer called paraganglioma.
  • Alternative names

    • SDH
    • Succinate coenzyme Q reductase
    • Succinate dehydrogenase
  • Database links

    • Entrez Gene: 6390 Human
    • Entrez Gene: 6389 Human
    • Entrez Gene: 6391 Human
    • SwissProt: P31040 Human
    • SwissProt: P21912 Human
    • SwissProt: Q99643 Human
    • SwissProt: O14521 Human
    • SwissProt: 6392 Human

    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

    • - 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.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (96)

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

    ab109908 has been referenced in 96 publications.

    • Rajab BS  et al. Differential remodelling of mitochondrial subpopulations and mitochondrial dysfunction are a feature of early stage diabetes. Sci Rep 12:978 (2022). PubMed: 35046471
    • Han G  et al. Dihuang-Yinzi Alleviates Cognition Deficits via Targeting Energy-Related Metabolism in an Alzheimer Mouse Model as Demonstrated by Integration of Metabolomics and Network Pharmacology. Front Aging Neurosci 14:873929 (2022). PubMed: 35431901
    • Erdem A  et al. Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability. Nat Commun 13:2013 (2022). PubMed: 35440568
    • Dong XQ  et al. Implication of a novel truncating mutation in titin as a cause of autosomal dominant left ventricular noncompaction. J Geriatr Cardiol 19:301-314 (2022). PubMed: 35572216
    • Zou R  et al. Empagliflozin attenuates cardiac microvascular ischemia/reperfusion injury through improving mitochondrial homeostasis. Cardiovasc Diabetol 21:106 (2022). PubMed: 35705980
    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 53 Abreviews or Q&A

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

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    abreview image
    Easy to follow instruction and standardize using cell lysate. Good sensitivity with microplate 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 27 2018

    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
    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.
    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.

    Dr. Amit Agarwal

    Verified customer

    Submitted Jul 24 2013

    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?

    Read More

    Abcam community

    Verified customer

    Asked on Aug 15 2012

    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.

    Read More

    Abcam Scientific Support

    Answered on Aug 15 2012

    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)?

    Read More

    Abcam community

    Verified customer

    Asked on Jan 31 2012

    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.

    Read More

    Abcam Scientific Support

    Answered on Jan 31 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

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

    Read More

    Abcam community

    Verified customer

    Asked on Apr 14 2014

    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!

    Read More

    Heather Allen

    Abcam Scientific Support

    Answered on Apr 14 2014

    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!

    Read More

    Abcam community

    Verified customer

    Asked on Feb 17 2014

    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.

    -----------------------------------------------------------------------------------------------

    Do you wish running an ELISA was fast and easy? Check out our new SimpleStep™ ELISA, a single wash, colorimetric sandwich ELISA assay.

    Discover more at www.abcam.com/simplestep

    Read More

    Heather Allen

    Abcam Scientific Support

    Answered on Feb 17 2014

    Question

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

    Read More

    Abcam community

    Verified customer

    Asked on Nov 20 2013

    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.

    Read More

    Padamjeet Singh

    Abcam Scientific Support

    Answered on Nov 20 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.

    Read More

    Abcam community

    Verified customer

    Asked on Jul 12 2013

    Answer

    The DCIP concentration is 2.5mM.

    Read More

    Keith B

    Abcam Scientific Support

    Answered on Jul 12 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

    1-10 of 53 Abreviews or Q&A

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