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
Overview
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Product name
NADP/NADPH Assay Kit
See all NADP/NADPH kits -
Detection method
Colorimetric -
Sample type
Tissue Extracts, Cell Lysate -
Assay type
Quantitative -
Assay time
2h 00m -
Product overview
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
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Notes
If you would like to use a fluorometric reading, please refer to NADP/NADPH Assay Kit (Fluorometric) (ab176724).
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. -
Platform
Microplate reader
Properties
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Storage instructions
Store 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 -
Research areas
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Relevance
NADP (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.
Images
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Functional studies- ab65349 Image from Hongxing Y et al., PLoS One 11(10), Fig 2a. Doi:10.1371/journal.pone.0164285 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
NAPDH/NADP+ ratio was determined using ab65349 in stable HSPB1- knockdown U87 cells.
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Functional assay: NADP/NADPH Assay Kit (ab65349)Standard curve with background signal subtracted (duplicates; +/- SD).
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Functional assay: NADP/NADPH Assay Kit (ab65349)Total NADP and NADPH (tNADP) or NADPH alone measured in RAW cell lysates (duplicates +/- SD).
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Functional Studies - NADP/NADPH Assay Kit (ab65349)Measurement of NADPt and NADPH in rat liver lysate (20 μg). Assays were performed using the kit protocol.
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Functional Assay - ab65349Measurement of NADPt and NADPH in HeLa cell lysate (80 μg). Assays were performed using the kit protocol.
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Typical Standard Curve - ab65349Typical standard curve for ab65349.
