NADP/NADPH Assay Kit (ab65349)
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.
Datasheets and documents
ab65349 has been referenced in 155 publications.
- Lin J et al. The POU2F1-ALDOA axis promotes the proliferation and chemoresistance of colon cancer cells by enhancing glycolysis and the pentose phosphate pathway activity. Oncogene 41:1024-1039 (2022). PubMed: 34997215
- Alaqbi SS et al. Increased mitochondrial proline metabolism sustains proliferation and survival of colorectal cancer cells. PLoS One 17:e0262364 (2022). PubMed: 35130302
- Pu F et al. LncCCAT1 interaction protein PKM2 upregulates SREBP2 phosphorylation to promote osteosarcoma tumorigenesis by enhancing the Warburg effect and lipogenesis. Int J Oncol 60:N/A (2022). PubMed: 35244192
- Zhang Y et al. Long noncoding RNA NEAT1 promotes ferroptosis by modulating the miR-362-3p/MIOX axis as a ceRNA. Cell Death Differ 29:1850-1863 (2022). PubMed: 35338333
- Yang Y et al. The Activation of AMPK/NRF2 Pathway in Lung Epithelial Cells Is Involved in the Protective Effects of Kinsenoside on Lipopolysaccharide-Induced Acute Lung Injury. Oxid Med Cell Longev 2022:3589277 (2022). PubMed: 35340214