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.
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1,4-Dihydronicotinamide adenine dinucleotide
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.
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.
How the assay works
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.
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.
NAD / NADH assay protocol summary
How other researchers are using NAD/NADH Assay Kit ab65348
This NAD/NADH assay kit has been used in publications in a variety of sample types, including:
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
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
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.
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).
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)
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.
Diagram showing the principles of the NAD NADH assay method.
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