Description
Key facts
Detection method:Colorimetric,
Sample types:Tissue, Cell culture supernatant, Cell Lysate,
Assay type:Quantitative,
Sensitivity:> 3 µM,
Range:3.13 - 100 µM,
Assay time:2h,
Assay Platform:Microplate reader
Product details:
NADH and NADPH Assay Kit (ab186031) provides a convenient method for detecting NADH and NADPH.
How the NADH / NADPH assay works
Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+. NAD forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. The traditional NAD/NADH and NADP/NADPH assays are based on monitoring the changes in NADH or NADPH absorption at 340 nm. The short UV wavelength of NAD/NADH and NADP/NADPH assays makes the traditional methods suffer low sensitivity and high interference.
In this NADH and NADPH assay method, the NADH/NADPH probe is a chromogenic sensor that has its maximum absorbance at 460 nm upon NADH/NADPH reduction. The absorbance maximum increases to ~ 635 nm if the enhancer is added to the assay system. The absorption of the NADH/NADPH probe is directly proportional to the concentration of NADH/NADPH in the solution.
Note that this assay kit will measure both NADPH and NADH together, and cannot differentiate them.
The Colorimetric NADPH Assay Kit provides a sensitive assay to detect as little as 3 µM NADPH in a 100 µL assay volume.
NADH and NADPH assay protocol summary
- prepare NADPH Reaction Mixture
- add NADPH standards or test samples
- incubate at room temperature for 15-120 minutes
- monitor Absorbance at 460 nm
REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
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.
NADH and NADPH along with their corresponding oxidized forms NAD+ and NADP+ function as critical coenzymes in cellular metabolism. These molecules serve as electron carriers in various biochemical reactions. NADH is primarily involved in energy production through the electron transport chain while NADPH functions mainly in reductive biosynthesis and antioxidant defense. Nadph has a molar mass of approximately 745.41 g/mol. Both are expressed in virtually all living cells due to their role in fundamental cellular processes.
Biological function summary
These coenzymes play distinct and integral roles in cellular metabolism. NADH primarily supports ATP generation by donating electrons to the mitochondria which culminate in the production of adenosine triphosphate (ATP). Conversely NADPH is essential in biosynthetic processes such as fatty acid and nucleic acid synthesis and it provides reducing power to counteract oxidative stress. These functions illustrate the importance of NADH and NADPH in maintaining cellular homeostasis and supporting anabolic reactions.
Pathways
NADH and NADPH are key participants in several vital pathways. In cellular respiration NADH is a central actor in glycolysis and the citric acid cycle where it facilitates the transfer of electrons to the electron transport chain enhancing oxidative phosphorylation. On the other hand NADPH serves a predominant role in the pentose phosphate pathway where it delivers reducing power for fatty acid synthesis and the reduction of glutathione. These pathways highlight their interplay with enzymes such as glucose-6-phosphate dehydrogenase and complex I of the mitochondria.
Disruptions in NADH and NADPH homeostasis can lead to various conditions including cancer and neurodegenerative diseases. Cancer cells often exhibit altered glucose metabolism known as the Warburg effect where NADPH supports increased biosynthesis and redox balance. Furthermore neurodegenerative diseases like Parkinson's are linked to mitochondrial dysfunction and oxidative stress where NADH and NADPH levels may become dysregulated. The sirtuin family of proteins which rely on NAD+ availability also highlight a connection between these coenzymes and disease pathophysiology.
