Description
Key facts
Detection method:Colorimetric,
Sample types:Tissue Extracts, Cell Lysate,
Assay type:Quantitative,
Assay time:2h,
Assay Platform:Microplate reader
Product details:
NADP/NADPH Assay Kit (ab65349) provides a convenient tool for sensitive detection of the intracellular nucleotides: NADP, NADPH and their ratio. Assays of nicotinamide nucleotides are of continual interest in the studies of energy transforming and redox state of cells or tissue.
The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction. The assay does not recognize NAD+/NADH. There is no need to purify NADP/NADPH from the sample mix. The enzyme cycling reaction significantly increases detection sensitivity. Results can be quantified using a plate reader at OD450nm.
NADP / NADPH assay protocol summary:
- extract samples from cells / tissues with extraction buffer and deproteinize with spin column
- for NADPH measurement, heat samples to 60°C for 30 min to decompose NAD
, cool on ice (this step not necessary for measurement of total NADP
/NADPH)
- add samples and standards to wells
- add reaction mix and incubate for 5 min at room temp to convert NADP to NADPH
- add NADPH 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
This product is manufactured by BioVision, an Abcam company and was previously called K347 NADP/NADPH Quantitation Colorimetric Kit. K347-100 is the same size as the 100 test size of ab65349.
If you would like to use a fluorometric reading, please refer to NADP/NADPH Assay Kit (Fluorometric) (ab176724)
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.
NADP/NADPH also known as Nicotinamide Adenine Dinucleotide Phosphate and its reduced form plays an essential role in cellular metabolism. It is a coenzyme with a molecular mass of around 743.4 g/mol. NADP/NADPH is ubiquitously expressed in cells due to its integral role in metabolic processes. It is found in high concentrations within the cytoplasm and chloroplasts where it functions in various biochemical reactions.
Biological function summary
NADPH facilitates reductive biosynthesis and maintains cellular redox status. It acts as a major electron donor in various anabolic reactions including lipid and nucleic acid synthesis. NADPH also contributes to the detoxification of reactive oxygen species and serves in the process of oxidative phosphorylation. Its cofactor activity associates it predominantly with enzymatic complexes such as the cytochrome P450 system.
Pathways
NADPH is fundamental in both the pentose phosphate pathway and the Calvin cycle. In the pentose phosphate pathway it provides reducing power important for fatty acid and cholesterol biosynthesis. In photosynthetic organisms NADPH functions in the Calvin cycle facilitating carbon fixation. The enzyme glucose-6-phosphate dehydrogenase related to NADPH production is integral in NADPH recycling.
Impaired NADPH function links to conditions like chronic granulomatous disease and diabetes. Chronic granulomatous disease occurs due to defects in the NADPH oxidase complex impairing the body’s ability to combat pathogens effectively. Diabetes has associations with oxidative stress influenced by imbalance in NADPH levels. In these diseases NADPH's interaction with proteins like superoxide dismutase affects oxidative damage management and overall immune response.
