Abcam, ab65348, NAD/NADH Assay Kit (Colorimetric)

Size: 100Test / 2000Test
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.
Key facts
Detection method:Colorimetric,
Sample types:Tissue Lysate, Urine, Serum, Cell Lysate,
Assay type:Quantitative,
Range:400 - 2000 nM,
Assay time:2h,
Assay Platform:Microplate reader

Product details:
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) (
ab176723
). NAD/NADH Assay kit
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
- 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
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:
- Human: primary blood mononuclear cells
, epithelial ovarian cancer cells
, Jurkat cells
- Mouse: cell culture lysates
, cardiomyocyte cell culture lysates
, liver
, liver and muscle
, primary hepatocyte cell cultures
, aorta tissue
- Rat: brain tissue
- Locust: thoraic muscle
Bacteria: Z mobilis
, E coli
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.

Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C

Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
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.
Biological function summary
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.
Pathways
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.