Overview

  • Product name

    Malate Dehydrogenase 2 (MDH2) Activity Assay
  • 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

Images

  • 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).
  • Figure 4. MDH2 antibody showing reactivity in a mitochondrial intracellular pattern with immunofluorescence microscopy.
  • Figure 2. MDH2 activity measurements of serially diluted human liver homogenate, rat heart homogenate, and mouse liver homogenate.
  • Figure 1. MDH2 activity measurements of serially diluted cultured HepG2 cell extracts.

Protocols

References

This product has been referenced in:

  • 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). Read more (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). Read more (PubMed: 27989324) »
See all 3 Publications for this product

Customer reviews and Q&As

1-10 of 10 Q&A

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

Answer



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

Read More

Question
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

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

Question
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

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

Question
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

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

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

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

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up