Key features and details
- Assay type: Quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
Product nameNADP/NADPH Assay Kit
See all NADP/NADPH kits
Sample typeTissue Extracts, Cell Lysate
Assay time2h 00m
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 60ºC 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).
Storage instructionsStore at -20°C. Please refer to protocols.
Components Identifier 100 tests NADP Cycling Buffer WM 1 x 15ml NADP Cycling Enzyme Mix Green 1 x 0.2ml NADP/NADPH Extraction Buffer NM 1 x 50ml NADPH Developer Purple 1 vial NADPH Standard (MW:833.36) Yellow 1 x 166.7µg Stop Solution Red 1 x 1.2ml
RelevanceNADP (Nicotinamide adenine dinucleotide phosphate) is a coenzyme composed of ribosylnicotinamide 5-phosphate (NMN) coupled by pyrophosphate linkage to the 5-phosphate adenosine 2,5-biphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidised (NADP+) and reduced (NADPH). The oxidative phase of the pentose phosphate pathway is the major source of NADPH in cells, producing approxiamtely 60% of the NADPH required. NADPH provides the reducing equivalents for biosynthetic reactions and the oxidation-reduction involved in protecting against the toxicity of ROS, allowing the regeneration of GSH. NADPH is also used for anabolic pathways, such as lipid synthesis, cholesterol synthesis and fatty acid chain elongation.
NAPDH/NADP+ ratio was determined using ab65349 in stable HSPB1- knockdown U87 cells.
Standard curve with background signal subtracted (duplicates; +/- SD).
Total NADP and NADPH (tNADP) or NADPH alone measured in RAW cell lysates (duplicates +/- SD).
Measurement of NADPt and NADPH in rat liver lysate (20 μg). Assays were performed using the kit protocol.
Measurement of NADPt and NADPH in HeLa cell lysate (80 μg). Assays were performed using the kit protocol.
Typical standard curve for ab65349.
ab65349 has been referenced in 82 publications.
- Tian W et al. miR-218 inhibits glucose metabolism in non-small cell lung cancer via the NF-?B signaling pathway. Exp Ther Med 21:106 (2021). PubMed: 33335569
- Ch R et al. Rhythmic glucose metabolism regulates the redox circadian clockwork in human red blood cells. Nat Commun 12:377 (2021). PubMed: 33452240
- Gong J et al. The pentose phosphate pathway mediates hyperoxia-induced lung vascular dysgenesis and alveolar simplification in neonates. JCI Insight 6:N/A (2021). PubMed: 33497360
- Tsai KL et al. Anti-IL-20 Antibody Protects against Ischemia/Reperfusion-Impaired Myocardial Function through Modulation of Oxidative Injuries, Inflammation and Cardiac Remodeling. Antioxidants (Basel) 10:N/A (2021). PubMed: 33578994
- Li Q et al. MALAT1 sponges miR-26a and miR-26b to regulate endothelial cell angiogenesis via PFKFB3-driven glycolysis in early-onset preeclampsia. Mol Ther Nucleic Acids 23:897-907 (2021). PubMed: 33614238