Overview

  • Product name

    Total NADP and NADPH Assay Kit (Colorimetric)
    See all NADP/NADPH kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant
  • Assay type

    Quantitative
  • Sensitivity

    > 0.03 µM
  • Range

    0.031 µM - 1 µM
  • Assay time

    2h 00m
  • Product overview

    Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+. NAD forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. The traditional NAD/NADH and NADP/NADPH assays are based on monitoring the changes in NADH or NADPH absorption at 340 nm. The short UV wavelength of NAD/NADH and NADP/NADPH assays makes the traditional methods suffer low sensitivity and high interference.

    Abcam’s Colorimetric total NADP/NADPH Assay Kit (ab136033) provides a convenient method for detecting total NADP and NADPH. The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction. There is no need to purify NADP/NADPH from the sample mix. The enzyme cycling reaction significantly increases detection sensitivity. The NADPH probe is a chromogenic sensor that has its maximum absorbance at 460 nm upon NADH reduction. The absorbance maximum increases to ~ 635 nm if the enhancer is added to the assay system. The absorption of the NADPH probe is directly proportional to the concentration of NADPH in the solution. The Colorimetric total NADP and NADPH Assay Kit provides a sensitive assay to detect as little as 0.03 µM total NADP/NADPH in a 100 µL assay volume.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 400 tests
    Lysis Buffer 1 x 10ml
    NADP/NADPH Recycling Enzyme Mixture 2 vials
    NADPH Probe 1 x 4ml
    NADPH Probe Buffer 1 x 16ml
    NADPH Standard 1 x 167µg
  • Research areas

  • 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

  • NADP/NADPH measured in mouse tissue lysates (5-50 mg/mL tested, expressed as quantity per mg of extracted protein; duplicates +/- SD).

  • NADP/NADPH measured cell lysates, where 5e6 cells were lysed in 0.25 mL. RAW samples were diluted 1/25-1/125 times, Jurkat samples were below level of detection (duplicates +/- SD).

  • NADP/NADPH measured in undiluted human biological fluids (duplicates +/- SD).

  • Standard curve with background signal subtracted (duplicates; +/- SD).

  • NADPH dose response was measured with the Colorimetric Total NADP/NADPH Assay Kit in a 96-well white/clear bottom plate using a microplate reader. As low as 0.03 µM of NADPH can be detected with 1 hour incubation (n=3) with absorbance measurement at 460nm.

    The absorbance in blank wells (with the PBS buffer only) is used as a control, and is subtracted from the values for those wells with the NADPH reactions.

     

     

Protocols

References

This product has been referenced in:

See 1 Publication for this product

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