NAD/NADH Assay Kit (Colorimetric) (ab65348)

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Overview

  • Product name
    NAD/NADH Assay Kit (Colorimetric)
    See all NAD/NADH kits
  • Detection method
    Colorimetric
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay type
    Quantitative
  • Assay time
    2h 00m
  • Product overview

    NAD/NADH Assay Kit (Colorimetric) ab65348 provides a convenient and sensitive tool to quantify NAD+ and NADH, and measure their ratio, in samples from mammals and other species.


    The NAD cycling enzyme mix in the kit specifically acts on NADH/NAD in an enzyme cycling reaction which significantly increases sensitivity and specificity. There is no requirement to purify NADH/NAD from samples.


    The levels of both NADt (total NAD+ and NADH) and NADH can be easily measured; the level of NAD+ can be easily calculated by subtracting NADH from NADt.


    NAD / NADH assay protocol summary:
    - extract samples from cells /  tissues with extraction buffer and deproteinize with spin column
    - for NADH measurement, heat samples to 60ºC for 30 min to decompose NAD+, cool on ice (this step not necessary for measurement of total NAD+/NADH)
    - add samples and standards to wells
    - add reaction mix and incubate for 5 min at room temp to convert NAD to NADH
    - add NADH developer and incubate for 1-4 hrs while reaction cycles
    - analyze with microplate reader multiple times during the 1-4 hr incubation
    - reaction can be stopped with stop solution

  • Notes

    This assay specifically detects NAD and NADH, but not NADP nor NADPH.

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

    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.

  • Platform
    Microplate reader

Properties

  • 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

Images

  • Functional Studies - NAD/NADH Assay Kit (Colorimetric) (ab65348)
    Functional Studies - NAD/NADH Assay Kit (Colorimetric) (ab65348)Karandrea, Shpetim et al., PloS one vol. 12,9 e0185374., Fig 6, doi:10.1371/journal.pone.0185374

    NADH/NAD+ ratio in soleus muscle measured useing ab65348. p ? 0.05 when comparing (+) HFD and LFD, (*) HFD + TQ and HFD, and (#) LFD and LFD + TQ using independent t-tests. Results are means ± SEM (n = 8-10 mice per treatment group). LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.

  • Functional Studies - NAD/NADH Assay Kit (ab65348)
    Functional Studies - NAD/NADH Assay Kit (ab65348)
    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)

  • Functional assay: NAD/NADH Assay Kit (ab65348)
    Functional assay: NAD/NADH Assay Kit (ab65348)

    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).

  • Functional assay: NAD/NADH Assay Kit (ab65348)
    Functional assay: NAD/NADH Assay Kit (ab65348)

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

  • Functional Studies - NAD+/NADH Assay Kit (ab65348)
    Functional Studies - NAD+/NADH Assay Kit (ab65348)
    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.

Protocols

References

This product has been referenced in:
  • Mekala NK  et al. Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue. Redox Biol 20:107-117 (2019). Read more (PubMed: 30300862) »
  • Ding D  et al. Genetic variation in PTPN1 contributes to metabolic adaptation to high-altitude hypoxia in Tibetan migratory locusts. Nat Commun 9:4991 (2018). Read more (PubMed: 30478313) »
See all 40 Publications for this product

Publishing research using ab65348? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question

Is the extraction buffer the same for both these kits?

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Answer

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

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Question

Could you please provide the range to measure nM for NAD/NADH Assay Kit (ab65348).

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Answer

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.

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Question

Can adherent cells be extracted in the plate, rather than removing them by trypsinization or other method and pelleting?

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Answer

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. If you have a rubber scraper, you might want to scrape the monolayers off the well bottoms after the first thaw, depending on how the cells look (intact or lysed). Identifying lysed cells will be difficult without a trypan blue stain though, so if you have a spare well you could check with that before proceeding.

Trypsinization before freeze/thaw might lead to more complete lysis of the entire population but we do not have data from a comparison and the difference will probably vary among cell lines. If you have a few extra replicate wells, you could try this yourself and compare the efficiency of trypsinization followed by 2X freeze/ thaw. The question is whether trypsinization of metabollically active cells will shift NAD/NADH ratios, perhaps obscuring differences within the experiment.

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Question

1) How can I avoid the foaminess when the NAD cycling enzyme is redissolved in the NAD cycling buffer (p.7 of protocol)? I used the pipette slowly up and down, but still ˜ 40ul of the 220 ul were lost and I ended up getting way less aliquots than expected and now as I result I don't have enough enzyme to perform the 100 assays.

2) Why does heat leads to NAD decomposition but the NADH is not affected (p.10 in protocol)?

3) After adding the stopping solution it is recommended to seal the plate. Is that wrapping with parafilm or just closing it (p.14)?

4) On p.12 of the protocol where a set up of the plate is presented, it seems like in the stnds, 50 ul of the master mix are added, BUT then in the protocol p. 13, each reaction requires ˜100 ul of master mix.

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Answer



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.


3) Seal the plate with a sticky transparent sheet that comes in the exact size as the plate.


4) 100 uL of master mix should be added to each well, and 50 uL of standard solution. The black text on the plate chart corresponds to the master mix, and the blue text corresponds to the standards. The green text corresponds to the samples.

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Question

I have purchased the NAD/NADH Assay Kit (ab65348), and I have a question about the protocol. Step 5 says to “let the reaction cycling at room temperature for 1 to 4 hours or longer depending on the reading of OD 450 nm” How do I know when the reaction has proceeded long enough? What OD should I be looking for to indicate that sufficient time has elapsed? Then, step 6 says “The reactions can be stopped by adding 10 uL of Stop Solution into each well and mix well. The color should be stable within 48 hours…” To be clear, I let the reaction cycle until a certain OD is attained and then I add stop solution? Do I have to wait about 48 hours after the stop solution is added before reading the plate at 450 nm?

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Answer


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 taken within that time.

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Question

rat heart tissue samples
400 ug protein in 50 ul per well
OD450 nm read outs:
NADtotal NADH
1.344 1.36
1.355 1.444
baseline: 0.135
but NADH should have smaller values than NADtotal
followed protocol step by step
standard curve is fine, but it uses NADH only, not the converting enzyme

did not use spin columns to remove NADH degrading enzymes, but this does not seem to be the problem here

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Answer

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 contain some interfering agents which are responsible for the lower readings in the NADt samples. It is crucial to use the filtration procedure to overcome this issue (using the spin columns as mentioned in the protocol).
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Question

Developer and enzyme mix appearance.

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Answer

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

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Question

Is it possible to use more cells, or would you need to use a larger volume of Extraction Buffer if you were to do that?

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Answer



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 number of cells, it will not be possible to get a more concentrated sample.

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Question

We are using NADP/NADPH Assay Kit ab65349 for measuring NADPH in lymphoma
cells currently, but we also want to measure NADH in the same cells.
We are going to order NAD/NADH Assay Kit (ab65348)very soon.
Can we share the cell extracts, which processed with the Extraction Buffer
offered in NADP/NADPH kit, for the assay with the NAD/NADH Assay kit?

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Answer


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).

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Question

Product code: 65348

Inquiry: Is the NAD/NADH kit working with plant tissue? Thanks,

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Answer

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:

1. Comparison of Tissue Preparation Methods for Assay of ...www.plantphysiol.org/content/84/4/987.full.pdf
by Z Zhao - 1987 - Cited by 21 - Related articles
Mar 23, 1987 – ABSTRACT. To prepare tissues for analysis of NAD', NADH, NADPI, and... Recoveries and Levels of Coenzymes in Plants. Recoveries of ...

2.NAD +/NADH enzyme cycling assay in plants--HELP - Biochemistrywww.protocol-online.org/biology-forums/posts/20658.html
Sep 29, 2006 – NAD+/NADH enzyme cycling assay in plants--HELP - enzyme assays ...an assay to quantify NAD+/NADH levels in A. thaliana leaf tissue.

Once you have optimized out a way to prepare the sample by proper homogenization etc, this kit might be useful to assay NAD, in theory.

I am sorry this is not tested for exactly what you require on this occasion, but I hope the information will be helpful. If you have any further questions, please do not hesitate to contact us.

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