• Product name
    NAD/NADH Assay Kit (Colorimetric)
    See all NAD/NADH kits
  • Detection method
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay type
  • 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.

    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Notes

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

  • Tested applications
    Suitable for: Functional Studiesmore details
  • Platform


  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    NAD Cycling Buffer NM 1 x 15ml
    NAD Cycling Enzyme Mix Green 1 vial
    NADH Developer Purple 1 vial
    NADH Standard (MW:663.4) Yellow 1 x 200nmole
    NADH/NAD Extraction Buffer NM 1 x 50ml
    Stop Solution Red 1 x 1.2ml
  • Research areas
  • Relevance
    NAD (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 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)

  • 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).

  • 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.



This product has been referenced in:
  • Long YM  et al. Surface ligand controls silver ion release of nanosilver and its antibacterial activity against Escherichia coli. Int J Nanomedicine 12:3193-3206 (2017). Read more (PubMed: 28458540) »
  • Shao D  et al. Glutaredoxin-1 Deficiency Causes Fatty Liver and Dyslipidemia by Inhibiting Sirtuin-1. Antioxid Redox Signal 27:313-327 (2017). Read more (PubMed: 27958883) »

See all 34 Publications for this product

Customer reviews and Q&As

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

1-10 of 106 Abreviews or Q&A


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