Description
Key facts
Detection method:Colorimetric,
Sample types:Cell Lysate,
Assay type:Quantitative,
Sensitivity:= 62.5 nM,
Assay Platform:Microplate reader
Product details:
NAD/NADH Assay Kit II (Colorimetric) (ab221821) provides a sensitive and robust method to measure NAD
, NADH and their ratio in mammalian cell lysates. The assay is based on an ADH and diaphorase coupled-reaction that convers WST-1 to WST-1 formazan, which can be easily detected at OD 450 nm. As the reaction is not stopped, it is necessary to monitor the absorbance increase of WST-1 formazan at regular intervals after the reaction is initiated to determine the reaction velocity.
This assay requires purification of NAD
and NADH from the cell lysates, which raises the efficiency of the reaction and increases the detection sensitivity.
Nicotinamide nucleotides are key players in the energy and oxidation-reduction reactions of a cells. Nicotinamide adenine dinucleotide (NAD) exists in two forms, an oxidized form, NAD+, and a reduced form, NADH. NAD functions as a cofactor in the vast majority of cellular redox reactions, carrying reducing equivalents from one reaction to another. Therefore, maintaining appropriate levels of NAD is essential for maintaining normal cellular respiratory function. There are two major pathways in NAD biosynthesis. The
de novo
pathway is maintained by the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), whereas the savage pathway recycles degraded NAD products such as nicotinamide. Studies have shown that cytosolic NAD
concentrations range from 300 nM in mammalian cells to 2 mM in yeast. Depletion of NAD in cells is a major cause of cell death.
The importance of NAD function in modulating cellular redox status and controlling signaling and transcriptional events makes NAD an important cofactor when investigating normal cellular function.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--80°C, Appropriate long-term storage conditions--80°C, Storage information--80°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.
