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.
Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.
Colorimetric
Tissue Lysate, Urine, Serum, Cell Lysate
Quantitative
400 - 2000 nM
2h
Select an associated product type
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.
Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.
Colorimetric
Tissue Lysate, Urine, Serum, Cell Lysate
Quantitative
400 - 2000 nM
2h
Microplate reader
Blue Ice
-20°C
-20°C
-20°C
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. The assay is read by absorbance at 450 nm.
NAD / NADH assay protocol summary:
- extract samples from cells / tissues with extraction buffer and deproteinize with spin column
- for NADH measurement, heat samples to 60°C 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.
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) (NAD/NADH Assay Kit (Fluorometric) ab176723). NAD/NADH Assay kit NAD/NADH Assay Kit II (colorimetric) ab221821 uses an alternative assay method that relies on purification of NAD and NADH from samples and may be more sensitive in some samples. Review our 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. **How other researchers have used NAD/NADH Assay Kit ab65348** This NAD/NADH assay kit has been used in publications in a variety of sample types, including: - Human: primary blood mononuclear cells1, epithelial ovarian cancer cells2, Jurkat cells3 - Mouse: cell culture lysates4, cardiomyocyte cell culture lysates5, liver6, liver and muscle7, primary hepatocyte cell cultures8, aorta tissue9 - Rat: brain tissue10 - Locust: thoraic muscle11 Bacteria: Z mobilis12, E coli13 References: 1 - Castro-Marrero J et al 2015; 2 - Zhu J et al 2019, Xia H et al 2015; 3 - Miller TW et al 2015; 4 - Mekala NK et al 2019, Ling S et al 2017; 5 - Zhang D et al 2019; 6 - Shao D et al 2017, Mukherji A et al 2015, Yu JH et al 2016; 7 - Karandrea S et al 2017; 8 - Traboulsi H et al 2014; 9 - Liu Y et al 2016; 10 - Rao G et al 2016; 11 - Ding D et al 2018; 12 - Wu B et al 2019; 13 - Long YM et al 2017
NAD/NADH often called nicotinamide adenine dinucleotide acts as an important coenzyme in redox reactions. It has a molecular mass of 663.43 g/mol. NAD/NADH exists widely across all living cells functioning predominantly in the cytoplasm and mitochondria. It is an important component in metabolic pathways serving as an electron carrier in processes that generate and use ATP. This coenzyme oscillates between oxidized (NAD⁺) and reduced (NADH) forms essential for energy production.
The oscillation between NAD⁺ and NADH enables cells to maintain redox homeostasis. It is not part of a larger protein complex but plays an important role in several biological reactions by transferring electrons. NAD⁺ works as an electron acceptor while NADH serves as an electron donor. Their balance influences various cellular processes including DNA repair and gene expression regulation. This coenzyme is essential to the enzymatic activity of dehydrogenases which are pivotal for the energy metabolism.
NAD+/NADH balance is vital in glycolysis and the citric acid cycle. Glycolysis uses NAD⁺ to help break down glucose into pyruvate while the citric acid cycle further processes acetyl-CoA producing NADH. NADH produced in these pathways then enters the electron transport chain driving ATP synthesis. This coenzyme also connects with sirtuins a family of proteins known for regulating cellular health and longevity through NAD⁺-dependent deacetylase activity.
NAD/NADH imbalance links with metabolic conditions like diabetes and neurodegenerative diseases such as Alzheimer's. In diabetes altered NAD+/NADH ratio can impact insulin secretion and glucose metabolism. Alzheimer's disease associates with disrupted NAD+ levels that affect sirtuins' function leading to impaired cellular repair mechanisms. These connections illustrate the coenzyme's significant effect on human health and highlight its potential as a therapeutic target.
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Terms & Conditions.
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.
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. Standard curve range: 20-100 pmol.
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