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    products/assay-kits/malate-dehydrogenase-2-mdh2-activity-assay-ab119693.pdf

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Malate Dehydrogenase 2 (MDH2) Activity Assay (ab119693)

  • Datasheet
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  • Protocol Booklet
Submit a review Q&A (10)References (7)

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ELISA - MDH2 Human Activity Assay Kit (ab119693)
  • Immunocytochemistry/ Immunofluorescence - MDH2 Human Activity Assay Kit (ab119693)
  • ELISA - MDH2 Human Activity Assay Kit (ab119693)
  • ELISA - MDH2 Human Activity Assay Kit (ab119693)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Sample type: Cell culture extracts, Tissue Extracts
  • Sensitivity: 0.78 µg/ml

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Overview

  • Product name

    Malate Dehydrogenase 2 (MDH2) Activity Assay
    See all MDH2 kits
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    1 3 4.1%
    Inter-assay
    Sample n Mean SD CV%
    1 3 13.9%
  • Sample type

    Cell culture extracts, Tissue Extracts
  • Assay type

    Enzyme activity
  • Sensitivity

    0.78 µg/ml
  • Range

    0.78 µg/ml - 200 µg/ml
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    Malate Dehydrogenase 2 (MDH2) Activity Assay (ab119693) is used to determine mitochondrial malate dehydrogenase activity (MDH2) in a sample. The enzyme is captured within the wells of the microplate and activity is determined by following the production of NADH in the following MDH2 catalyzed reaction: malate + NAD → oxaloacetic acid + NADH (↑ Absorbance at 450 nm). The generation of NADH is coupled to the 1:1 reduction of a reporter dye to yield a colored (yellow) reaction product whose concentration can be monitored by measuring the increase in absorbance at 450 nm. In each well, ab119693 immunocaptures only native MDH2 from the chosen sample; this removes all other enzymes, including MDH1 in cytosol.


    This product allows researchers to focus on TCA cycle, studying isotype-specific malate dehydrogenase (MDH2) activity assay without the necessity of isolating mitochondria.

  • Notes

    Mitochondrial malate dehydrogenase (MDH2, P40926) is a 35.5 kDa enzyme that catalyzes the conversion of malate into oxaloacetate (using NAD+) and vice versa. (EC 1.1.1.37) Several isozymes of malate dehydrogenase exist, depending on where they are localized in the cell and their specific dependence on NAD+ or NADP+ (only in chloroplasts). There are two main isoforms in eukaryotic cells. One is found in the mitochondrial matrix (MDH2), participating as a key enzyme in the citric acid cycle that catalyzes the oxidation of malate. The other is found in the cytoplasm (MDH1), assisting the malate-aspartate shuttle with exchanging reducing equivalents so that malate can pass through the mitochondrial membrane to be transformed into oxaloacetate for further cellular processes. Because malate dehydrogenase is closely tied to the citric acid cycle, regulation is highly dependent on TCA products. High malate concentrations stimulate MDH activity, and, in a converse manner, high oxaloacetate concentrations inhibit the enzyme. Enzyme activity is enhanced by acetylation.

    Storage: All components are shipped cold. Reagent dye, coupler, malate and NAD+ are shipped lyophilized. Before use rehydrate by adding 0.25 mL pure H2O to each tube and vortex each tube thoroughly to dissolve. After hydration unused amounts of these four materials should be stored at -80°C for 6 months. Store all other components at 4°C. This kit is stable for 6 months from receipt.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    100X Coupler 1 unit
    100X NAD+ 1 unit
    100X Reagent Dye 1 unit
    100X Sodium Malate 1 unit
    10X Blocking Buffer 1 x 8ml
    20X Buffer 1 x 20ml
    Base Buffer 1 x 24ml
    Extraction Buffer (ab260490) 1 x 15ml
    MDH2 Microplate 1 unit
  • Research areas

    • Tags & Cell Markers
    • Subcellular Markers
    • Organelles
    • Mitochondria
    • Signal Transduction
    • Metabolism
    • Energy Metabolism
    • Signal Transduction
    • Metabolism
    • Mitochondrial
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Dehydrogenase Kits
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Mitochondrial markers
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Energy transfer pathways
    • Energy Metabolism
  • Sequence similarities

    Belongs to the LDH/MDH superfamily. MDH type 1 family.
  • Post-translational
    modifications

    Acetylation is enhanced by up to 67% after treatment either with trichostin A (TSA) or with nicotinamide (NAM) with the appearance of tri-and tetraacetylations. Glucose also increases acetylation by about 60%.
  • Cellular localization

    Mitochondrion matrix.
  • Target information above from: UniProt accession P40926 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • M MDH
    • Malate dehydrogenase
    • Malate dehydrogenase 2, NAD (mitochondrial)
    • Malate dehydrogenase, mitochondrial
    • MDH
    • mdh2
    • MDHM_HUMAN
    • MGC:3559
    • mitochondrial
    • Mitochondrial malate dehydrogenase 2, NAD
    • Mor 1
    • MOR1
    see all
  • Database links

    • Entrez Gene: 4191 Human
    • Entrez Gene: 17448 Mouse
    • Entrez Gene: 81829 Rat
    • Omim: 154100 Human
    • SwissProt: P40926 Human
    • SwissProt: P08249 Mouse
    • SwissProt: P04636 Rat
    • Unigene: 520967 Human
    • Unigene: 297096 Mouse
    • Unigene: 1011 Rat
    see all

Associated products

  • Positive Controls

    • 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

  • ELISA - MDH2 Human Activity Assay Kit (ab119693)
    ELISA - MDH2 Human Activity Assay Kit (ab119693)
    Figures 7. The isoform specificity of the malate activity measured by this kit is confirmed by measuring the MDH activity from different cell fractions. Activity was only detected from the mitochondrial fraction (MDH2), not the cytosol fraction (MDH1).
  • Immunocytochemistry/ Immunofluorescence - MDH2 Human Activity Assay Kit (ab119693)
    Immunocytochemistry/ Immunofluorescence - MDH2 Human Activity Assay Kit (ab119693)
    Figure 4. MDH2 antibody showing reactivity in a mitochondrial intracellular pattern with immunofluorescence microscopy.
  • ELISA - MDH2 Human Activity Assay Kit (ab119693)
    ELISA - MDH2 Human Activity Assay Kit (ab119693)
    Figure 2. MDH2 activity measurements of serially diluted human liver homogenate, rat heart homogenate, and mouse liver homogenate.
  • ELISA - MDH2 Human Activity Assay Kit (ab119693)
    ELISA - MDH2 Human Activity Assay Kit (ab119693)
    Figure 1. MDH2 activity measurements of serially diluted cultured HepG2 cell extracts.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (7)

Publishing research using ab119693? Please let us know so that we can cite the reference in this datasheet.

ab119693 has been referenced in 7 publications.

  • Guo X  et al. The Role of Palmitoleic Acid in Regulating Hepatic Gluconeogenesis through SIRT3 in Obese Mice. Nutrients 14:N/A (2022). PubMed: 35406095
  • Barbier-Torres L  et al. Silencing hepatic MCJ attenuates non-alcoholic fatty liver disease (NAFLD) by increasing mitochondrial fatty acid oxidation. Nat Commun 11:3360 (2020). PubMed: 32620763
  • Jones CL  et al. Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells. Cell Stem Cell 27:748-764.e4 (2020). PubMed: 32822582
  • Calmels C  et al. Application of a genome-scale model in tandem with enzyme assays for identification of metabolic signatures of high and low CHO cell producers. Metab Eng Commun 9:e00097 (2019). PubMed: 31720213
  • Lee WT  et al. Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns. Cell Death Discov 3:17062 (2017). PubMed: 28900542
  • Ait-El-Mkadem S  et al. Mutations in MDH2, Encoding a Krebs Cycle Enzyme, Cause Early-Onset Severe Encephalopathy. Am J Hum Genet 100:151-159 (2017). PubMed: 27989324
  • Kim EY  et al. Acceleration of adipogenic differentiation via acetylation of malate dehydrogenase 2. Biochem Biophys Res Commun 441:77-82 (2013). PubMed: 24134846

Customer reviews and Q&As

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

1-10 of 10 Abreviews or Q&A

Question

I was wondering if you could tell me where exactly on MDH2 your anti MDH2 antibody ab110317 (clone #2F5AF8) binds? I am also assuming this is the antibody that is used to coat the assay plate wells in your MDH2 activity assay kit ab119693?


We have recently bought in a couple of these assay kits to use to test out some small molecule inhibitors (including the published and commercially available MDH2 inhibitor LW6). I have been isolating the MDH2 from my cell lysates first and then adding compound along with the activity solution but cannot seem to show any effect on enzymatic activity with the published compound. My lysates are fine and you can pull down active MDH2 which shows a nice dose responsive reaction rate with increasing lysate concentration. But for some reason I cannot inhibit this activity. I am wondering whether binding of MDH2 to the antibody could in some way be hindering binding of the inhibitor?

Read More

Abcam community

Verified customer

Asked on Jan 22 2014

Answer


You are right we use 2F5AF8 as the capture antibody to purify MDH2 from samples. The immunogen of the antibody is purified MDH2, we do not know the epitope of this antibody, but we know the antibody did not bind to the active site, substrate binding site or NAD binding site.

In your case, make sure you add the inhibitor to the reaction buffer and mix well, then add the (reaction buffer + inhibitor) to the well, which contains isolated MDH2.

The antibody binding should not interfere with the inhibition since you inhibitor should bind to either active site, substrate binding site or NAD binding site.

We have not tested the recombinant MDH2 proteins or LW6 inhibitor. But following literature prove it works. The paper is published using Abcam MDH2 activity assay kit.

http://onlinelibrary.wiley.com/doi/10.1002/anie.201304987/pdf;
supplemental data: http://onlinelibrary.wiley.com/store/10.1002/anie.201304987/asset/supinfo/anie_201304987_sm_miscellaneous_information.pdf?)v=1&s=87fa29e8195198e8da493ff30fd08a935f623268


Read More

Abcam Scientific Support

Answered on Jan 22 2014

Question

I was planning on flash freezing the livers in liquid nitrogen and storing at -80C until I decided on a kit. I was wondering if that form of storage would be compatible with using your MDH2 kit for mice.

Read More

Abcam community

Verified customer

Asked on Mar 14 2013

Answer



The MDH2 activity assay kit ab119693 is compatible with frozen tissue.

Read More

Abcam Scientific Support

Answered on Mar 14 2013

Question

Will this product work with other sample types, e.g. the purified protein?

Read More

Abcam community

Verified customer

Asked on Feb 06 2013

Answer

We would recommend dilution of the protein to a working range using 1X incubation buffer. If loading samples with more than 50% detergent, we suggest adding the same amount of detergent to the protein. Please have care when adding a recombinant protein, as many of these proteins are not active, particularly if expressed in E.coli.

Read More

Abcam Scientific Support

Answered on Feb 06 2013

Question

Hello, I need a malate assay for bacteria (legionella). Do you know if I could use some of yours products? Thanks

Read More

Abcam community

Verified customer

Asked on Dec 05 2012

Answer

Thank you for contacting us.

The product referred in your email (ab119693) is not intended to measure Malate in your samples, but the enzymatic activity of Malate dehydrogenase.

We do have a different kit in the catalogue, ab83391, which measures Malate in various sample types. Although we have not specifically tested it with bacteria, we predict it may work with this species.

I’d like to bring your attention to an offer we are running throughout November. If you order any primary antibody you can receive a RabMab free, whilst stocks last. Simply quote “RABMAB-XBSMG” in your next primary antibody order. More information on this offer can be found from the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=15447

I hope this helps. Otherwise do not hesitate to contact me back.

Read More

Abcam Scientific Support

Answered on Dec 05 2012

Question

I just need some more extraction buffer from this kit. Is that available separately?

Read More

Abcam community

Verified customer

Asked on Oct 30 2012

Answer

Our suggestion is to purchase 15% Lauryl Maltoside Solution (ab109858) and then produce the following buffer with the following recipe:


25 mM Hepes
100 mM NaCl
1.5% Lauryl Maltoside
pH 7.4

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

Read More

Abcam Scientific Support

Answered on Oct 30 2012

Question

I have a question about analyzing my data. In the protocol on p. 15, the y axis of the graph is change in mOD/min, but what was the change in OD that was used? The protocol says, "For simplicity the activity can be expressed as the change in absorbance/min/amount of sample loaded into well." How is this done?

Read More

Abcam community

Verified customer

Asked on Jun 29 2012

Answer

Thank you for your inquiry.

The lab was able to provide the following information:

The answer can be found at the beginning of the booklet: (page 3)
https://www.abcam.com/ps/products/119/ab119693/documents/ab119693%20%20MDH2%20Activity%20Booklet%20(Website).pdf
“ Principle: ab119693 is used to determine mitochondrial malate dehydrogenase activity (MDH2) in a sample. The enzyme is captured within the wells of the microplate and activity is determined by following the production of NADH in the following MDH2
catalyzed reaction:
malate + NAD+ _ oxaloacetic acid + NADH (_ Absorbance at 450 nm)
The generation of NADH is coupled to the 1:1 reduction of a reporter dye to yield a colored (yellow) reaction product whose concentration can be monitored by measuring the increase in absorbance at 450nm (Dye molar extinction coefficient - 37000 M-1 cm-1).”
I hope this information helps. Please let us know if you have any questions.

Read More

Abcam Scientific Support

Answered on Jun 29 2012

Question

Will flash frozen tissue workwith this kit?

Read More

Abcam community

Verified customer

Asked on Jun 08 2012

Answer

Thank you for contacting Abcam yesterday.

Yes, for activity assays the tissue section may be frozen. The tissue homogenate may also be frozen at -80C. What we don’t recommend is to freeze the extract after addition of the provided detergent.

Please let me know if there is anything else I can help you with.

Read More

Abcam Scientific Support

Answered on Jun 08 2012

Question

Thank you very much!



The amount is not a problem...I can have as many flies as I want!



I think I would like to try the kit....BUT how can I buy the kit without knowing if I can use tecan reader?!

Any idea? My boss wont approve buying it....without knowing we have the machine to read it:) LOL



Bw

Read More

Abcam community

Verified customer

Asked on Mar 23 2012

Answer

Thank you for your reply. My colleague Cathrin is not in the office today.

Unfortunately, I do also not know if this kit is compatible with a Tecan reader.

I can suggest to ask the manufacturer of the Tecan reader. They should be able to help you after they had a look at the datasheet of protocol of this kit.

I am sorry I could not be of more help and hope this information is nevertheless helpful.

Read More

Abcam Scientific Support

Answered on Mar 23 2012

Question

Do theywork on mice andDrosophilla? How specific is it? Tecan machine compatable (Flourescence)?Does the last buffer in the protocolincludes or need a detergent?

Read More

Abcam community

Verified customer

Asked on Mar 22 2012

Answer

Thank you for contacting us.

I have some good and some bad news for you. the good ones are that it will work in mice, and that the kits may work with Drosophila samples as we as the homology is for both enzymes compared with the human proteinsis over 98%. The bad one is that you will need quite an amount of material to get your experience running, and this may simply not possible with this test species.

These assays are specific to these enzymes and isoforms as demonstrated by mass spec analysis of immunoprecipitates.


Regarding the buffers, adetergent is not included in either final buffer.

And last but not least, unfortunately the lab couldn't tell me if your "Tecan reader" will work with the kits: they don't know it.



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 22 2012

Question

uses human skeletal muscle biopsies from children (i.e. has limited samples)
How much tissue do I need to startwith?
Is this different for each kit?

Read More

Abcam community

Verified customer

Asked on Feb 02 2012

Answer

Thank you for contacting us.

I have heard back from the lab with the following information:

All these assays require different sample concentrations described in each booklet protocol - but in most cases is approximately 10ug/microplate well.
The good news is that normal whole muscle homogenate contains a good robust mitochondria signal so less is required, muscle samples are described in most if not all protocols.
Another good news is that the same preparation for any of the assays is suitable for all others. I would estimate that if a 1 mg of homogenate can be prepared (0.2mL at 5mg/mL).
This would provide enough material for each of the assays when diluted to the specific concentration in each assay.
We would recommend - if contemplating doing multiple assays - to include a mitochondrial sample as positive control such as ab110338 isolated mitochondria (note this is cow source)



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 Feb 02 2012

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