NAD/NADH Assay Kit (Fluorometric) ab176723 provides a convenient method for sensitive detection of NAD, NADH and their ratio.
Colorimetric/Fluorometric
Tissue Lysate, Cell Lysate
Quantitative
Mammals
2h 30m
Select an associated product type
NAD/NADH Assay Kit (Fluorometric) ab176723 provides a convenient method for sensitive detection of NAD, NADH and their ratio.
Colorimetric/Fluorometric
Tissue Lysate, Cell Lysate
Quantitative
Mammals
2h 30m
Microplate reader
Blue Ice
-20°C
-20°C
-20°C
NAD/NADH Assay Kit (Fluorometric) ab176723 provides a convenient method for sensitive detection of NAD, NADH and their ratio.
The enzymes used in the NAD/NADH assay protocol specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the interference from biological samples.
There is no need to purify NAD/NADH from sample mix. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.
The NAD/NADH assay signal can be easily read by either a fluorescence microplate reader at Ex/Em 530 - 570/590 - 600 nm (max Ex/Em 540/590 nm) or an absorbance microplate reader at ~576 nm.
This kit provides NAD and NADH extraction buffer, and cell lysis buffer for your convenience. It has been frequently used for determining NAD/NADH from cell lysates.
NAD/NADH assay protocol summary:
- add standards and samples for NAD, NADH, total NAD/NADH measurement to wells
- add NADH extraction solution to NADH wells, incubate for 10-15 min, and add NAD extraction solution to neutralize
- add NAD extraction solution to NAD wells, incubate for 10-15 min, and add NADH extraction solution to neutralize
- add NAD/NADH control solution to standard and total NAD/NADH wells, incubate for 10-15 min, and add NAD/NADH control solution
- add NADH reaction mix and incubate for 30 min to 2 hr
- analyze with a microplate reader
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.
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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Total NADH and NAD Dose Response Curve.
Total NADH and NAD, and their extract dose response were measured with NAD/NADH Assay Kit (Fluorometric) (ab176723) in a 96-well black plate using a Gemini microplate reader (Molecular Devices). The signal was acquired at Ex/Em = 540/590 nm (cut off at 570 nm) 30 minutes after adding NAD/NADHH reaction mixture. The blank signal was subtracted from the values for those wells with the NADH reactions.
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