NADP/NADPH Assay Kit

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Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 2 hr
  • Sample type: Cell Lysate, Tissue Extracts

Overview

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
 

Notes

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).

Platform

Microplate reader

Properties

Storage instructions

Store at -20°C. Please refer to protocols.
ComponentsIdentifier100 tests
NADP Cycling BufferWM1 x 15ml
NADP Cycling Enzyme MixGreen1 x 0.2ml
NADP/NADPH Extraction BufferNM1 x 50ml
NADPH DeveloperPurple1 vial
NADPH Standard (MW:833.36)Yellow1 x 166.7µg
Stop SolutionRed1 x 1.2ml

Research areas

  • Kits/ Lysates/ Other
  •  
  • Kits
  •  
  • Cell Metabolism Kits
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  • Coenzyme and cofactor assay kits
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  • NADP/NADPH assay kits
  • Metabolism
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  • Pathways and Processes
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  • Cofactors, Vitamins / minerals
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  • Co-factors

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.

Protocols