• Product nameNAD/NADH Assay Kit (Colorimetric)
    See all NAD/NADH kits
  • Detection methodColorimetric
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay typeQuantitative
  • Assay time
    2h 00m
  • Species reactivity
    Reacts with: Mammal
  • Product overview

    NADH/NAD Quantification Kit (Colorimetric) (ab65348) provides a convenient tool for sensitive detection of the intracellular nucleotides: NADH, NAD and their ratio. Assay of nicotinamide nucleotides is of continual interest in the studies of energy transforming and redox state of cells or tissues.

    The NAD Cycling Enzyme Mix in the kit specifically recognizes NADH/NAD in an enzyme cycling reaction. There is no requirement to purify NADH/NAD from samples. The reaction specifically detects NAD and NADH, but not NADP nor NADPH. The enzyme cycling reaction significantly increases the detection sensitivity and specificity. NADt (NAD and NADH) or NADH can be easily quantified by comparing with standard NADH.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    If you would like to use a fluorometric reading, please refer to NAD/NADH Assay Kit (Fluorometric) (ab176723).

  • Tested applicationsSuitable for: Functional Studiesmore details
  • PlatformMicroplate


  • RelevanceNAD (Nicotinamide adenine dinucleotide) is a coenzyme in metabolic redox reactions, a precursor for several cell signaling molecules, and a substrate for protein posttranslational modifications. NAD is a dinucleotide, consisting of two nucleotides joined through their phosphate groups: with one nucleotide containing an adenosine ring, and the other containing nicotinamide. In metabolism, NAD is involved in redox reactions, carrying electrons from one reaction to another. The coenzyme is therefore found in two forms in cells: NAD is an oxidizing agent – it accepts electrons from other molecules and becomes reduced, forming NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD. However, it is also used in other cellular processes, the most notable one being a substrate of enzymes that add or remove chemical groups from proteins in posttranslational modifications.

Associated products


Our Abpromise guarantee covers the use of ab65348 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent dilution.

NAD/NADH Assay Kit (Colorimetric) images

  • NAD and NADH (tNAD) or NADH alone measured cell lysates. 5e6 cells were lysed in 1 mL, spin filtered, and tested neat or 1/5 (duplicates +/- SD).

  • Standard curve with background signal subtracted (duplicates; +/- SD).

  • NAD/NADH was measured in K562 ME2 knockdown cells(pLKO - empty vector; shME2-2 & shME2-3 - two selected knockdown clones). Data are expressed as mean ±: SD, n=3. NAD/NADH Ratio is calculated as described in the product protocol.

    Image obtained from Ren JG et al; PLOS one, 2010; 5(9): e12520 (DOI:10.1371/journal.pone.0012520)

  • NADH Standard calibration curve. Quantification of NAD (diamond) and NADH (open square) following product protocol and using NADH standard provided in the kit. No NADP (triangle) was detected in this reaction.


References for NAD/NADH Assay Kit (Colorimetric) (ab65348)

This product has been referenced in:
  • Yu JH  et al. Suppression of PPAR?-mediated monoacylglycerol O-acyltransferase 1 expression ameliorates alcoholic hepatic steatosis. Sci Rep 6:29352 (2016). Read more (PubMed: 27404390) »
  • Soldevila-Barreda JJ  et al. Transfer hydrogenation catalysis in cells as a new approach to anticancer drug design. Nat Commun 6:6582 (2015). Human . Read more (PubMed: 25791197) »

See all 25 Publications for this product

Product Wall

Yes, the extraction buffers from ab65348 and ab65349 are the same.

I can confirm that for ab65348 the detection range is 1-100 pmoles per well as shown on the STD curve (on-line product datasheet). The detection limit is 1 pmole in the sample volume added into the well.

Thank you for your question about how to assay adherent cells. The cells can be lysed by aspirating the medium and adding 400ul of the extraction buffer as we discussed, followed by 2 freeze-thaw cycles. That should be sufficient to lyse all the cells....

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1) To prevent extreme foaminess in the tube, do not add the 220ul of the NAD cycling buffer to the enzyme mix. Add a small volume at a time and wait.

2) NAD is much more heat-labile and hence it decomposes at 60C.

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Please take multiple readings during the 1-4 hr incubation and stop the reaction once the OD of the highest standard reaaches ˜1.9 or starts plateuing.

After adding the stop solution, OD readings will be stable for 48 hours and should be ...

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Thank you for contacting us.
The lab got back to me with the following advice and suggestion:
It seems that 400 µg/well is on the higher side. Please recommend 100-200 µg /well of the protein.
There is also a possibility that the samples c...

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I have confirmed with the lab that the developer is a red powder and the enzyme mix is a clear powder for ab65348.

It is definitely possible to use more cells to get a more concentrated sample. The volume of extraction buffer can be increased slightly, (for example, 500ul for 50,000 cells) but if you scale up the extraction buffer in the same ratio as the...

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We recommend using the buffers provided with the specific kits for our assays. So, unfortunately we don’t suggest using the same buffer for both assays (ab65348 and ab65349).

I am sorry to confirm this kit has not been specifically tested and guaranteed for use on plant samples.

The following references may provide information of preparing plant tissue for NAD and NADP measurement which I hope may be helpful: Read More

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