Overview

  • Product name

    Total NAD and NADH Assay Kit (Colorimetric)
    See all NAD/NADH kits
  • Detection method

    Colorimetric
  • Sample type

    Urine, Serum, Plasma, Cell Lysate, Tissue Lysate
  • Assay type

    Quantitative
  • Sensitivity

    > 0.1 µM
  • Range

    0.078 µM - 5 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 NAD/NADH Assay Kit (ab186032) provides a convenient method for detecting total NAD and NADH. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction. There is no need to purify NAD/NADH from the sample mix. The enzyme cycling reaction significantly increases detection sensitivity. The NADH probe is a chromogenic sensor that has its maximum absorbance at 460 nm upon NADH reduction. The absorption of the NADH probe is directly proportional to the concentration of NADH in the solution. The Colorimetric total NAD and NADH Assay Kit provides a sensitive assay to detect as little as 0.1 µM total NAD/NADH 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
    NAD/NADH Recycling Enzyme Mixture 2 vials
    NADH Probe 1 x 4ml
    NADH Probe Buffer 1 x 16ml
    NADH Standard 1 x 142µg
  • Research areas

  • Relevance

    NAD (Nicotinamide adenine dinucleotide) is a coenzyme in metabolic redox reactions, a precursor for several cell signaling molecules, and a substrate for protein posttranslational modifications. NAD is a dinucleotide, consisting of two nucleotides joined through their phosphate groups: with one nucleotide containing an adenosine ring, and the other containing nicotinamide. In metabolism, NAD is involved in redox reactions, carrying electrons from one reaction to another. The coenzyme is therefore found in two forms in cells: NAD is an oxidizing agent – it accepts electrons from other molecules and becomes reduced, forming NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD. However, it is also used in other cellular processes, the most notable one being a substrate of enzymes that add or remove chemical groups from proteins in posttranslational modifications.

Images

  • NAD/NADH measured in undiluted biological fluids (duplicates +/- SD).

  • NAD/NADH measured in mouse tissue lysates (1-20 mg protein x mL-1 tested, expressed as per mg of extracted protein; duplicates +/- SD).

  • NAD/NADH measured cell lysates (Jurkat sample below level of detection; duplicates +/- SD).

  • NADH dose response was measured with total NAD and NADH Assay Kit (Colorimetric)  in a 96-well white/clear bottom plate using a microplate reader. As low as 0.1 µM of NADH 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 NADH reactions.

     

Protocols

References

ab186032 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Answer


I contacted the originating laboratories. They say this kit has been tested externally for plasma and serum samples, but they recommend the following sample preparation protocols:

Serum: Collect blood without anticoagulant. Allow blood to clot for 30 minutes RT. Centrifuge at 2000 x g and 4ºC for 10 min. Remove serum layer and store on ice. Take care to avoid disturbing the white buffy layer. Aliquot samples for testing and store remaining solution at -80ºC. Prior to testing, filter samples with 3K-10K centrifugal filter. Perform serum dilutions in Assay Buffer.


Plasma: Collect blood with heparin or citrate and centrifuge at 1000 x g and 4ºC for 10 minutes. Remove the plasma layer and store on ice. Take care to avoid disturbing the white buffy layer. Aliquot samples for testing and store remaining solution at -80ºC. Perform plasma dilutions in Assay Buffer. Perform serum dilutions in Assay Buffer.

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