Malate Dehydrogenase 2 (MDH2) Activity Assay (ab119693)

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

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

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.

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.

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.

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.

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.

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.

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.

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.