Overview

  • Product name

    NAD/NADH Assay Kit (Fluorometric)
    See all NAD/NADH kits
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

    Cell Lysate, Tissue Lysate
  • Assay type

    Quantitative
  • Assay time

    2h 30m
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    NAD/NADH Assay Kit (Fluorometric) ab176723 provides a convenient method for sensitive detection of NAD, NADH and their ratio.


    The enzymes used in the NAD/NADH assay protocol specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the interference from biological samples.


    There is no need to purify NAD/NADH from sample mix. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.


    The NAD/NADH assay signal can be easily read by either a fluorescence microplate reader at Ex/Em 530 - 570/590 - 600 nm (max Ex/Em 540/590 nm) or an absorbance microplate reader at ~576 nm. 


    This kit provides NAD and NADH extraction buffer, and cell lysis buffer for your convenience. It has been frequently used for determining NAD/NADH from cell lysates. 


    NAD/NADH assay protocol summary:
    - add standards and samples for NAD, NADH, total NAD/NADH measurement to wells
    - add NADH extraction solution to NADH wells, incubate for 10-15 min, and add NAD extraction solution to neutralize
    - add NAD extraction solution to NAD wells, incubate for 10-15 min, and add NADH extraction solution to neutralize
    - add NAD/NADH control solution to standard and total NAD/NADH wells, incubate for 10-15 min, and add NAD/NADH control solution
    - add NADH reaction mix and incubate for 30 min to 2 hr
    - analyze with a microplate reader

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 250 tests
    NAD Extraction Solution 1 x 10ml
    NAD/NADH Control Solution 1 x 10ml
    NAD/NADH Lysis Buffer 1 x 10ml
    NAD/NADH Recycling Enzyme Mixture 2 vials
    NADH Extraction Solution 1 x 10ml
    NADH Sensor Buffer 1 x 20ml
    NADH Standard 1 vial
  • 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

  • Total NADH and NAD, and their extract dose response were measured with NAD/NADH Assay Kit (Fluorometric) (ab176723) in a 96-well black plate using a Gemini microplate reader (Molecular Devices). The signal was acquired at Ex/Em = 540/590 nm (cut off at 570 nm) 30 minutes after adding NAD/NADHH reaction mixture. The blank signal was subtracted from the values for those wells with the NADH reactions. 

Protocols

References

This product has been referenced in:

  • Zhang B  et al. Characterization of the Role of the Malate Dehydrogenases to Lung Tumor Cell Survival. J Cancer 8:2088-2096 (2017). Read more (PubMed: 28819410) »
See 1 Publication for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you for your reply.


No problem, the data analysis can be tricky for this kit.  I hope the following may help explain more clearly:


1. The reason there is no NAD standard curve:  Since NAD+ is converted to NADH in an enzyme cycling reaction and relatively more labile than NADH, only the NADH Standard curve is included.


2. The NADH is calculated as follows: Obtain the reading from the NADH extract, the NADH can be calculated from the NADH standard curve


3. To calculate NAD, minus the NADH calculated form the amount of total NAD/NADH


4. As an extra check for the amount of NAD, the NAD extract sample is included. In this sample, the NADH is decomposed by the extract buffer. The NAD left in the sample is then converted to NADH with a cycling enzyme. So when you measure the NADH, this should tell you how much NAD was originally in the sample.  (The dye used for detection works with NADH and does not work with NAD, hence why you need to convert to NADH).


I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

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NAD/NADH Assay Kit in C57 and mdx mice

Below average Good 4/5 (Ease of Use)
Abreviews
Consistent with the previous review, the total NAD/NADH was smaller than the NADH alone. In addition, the PBS which was used as a blank was large resulting in some negative values. Lastly, due to the small total NAD/NADH values data was expressed only as an NAD/NADH ratio. Contrary to our expectations, no significant differences were observed between C57 and mdx mice. This kit was attempted twice with similar results. There was no deviation from the protocol provided with the ki

Abcam user community

Verified customer

Submitted Jan 27 2016

NAD/NADH Assay Kit for mouse samples

Good Good 4/5 (Ease of Use)
Abreviews
You can find attached the results.
In summary the kit worked for mouse samples, but with some troubles during the procedure, regarding mainly the blank signal.

Abcam user community

Verified customer

Submitted Aug 07 2015

Answer

Thank you for your enquiry.
The enzymes in this kit are not luciferases, they are something like dehydrogenase which specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity.

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Answer

I can confirm that ab176723 can detect both free and protein bound NAD/NADH.

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Answer



Nous avons ajouté 1 flacon de NADH sensor buffer de 20 ml et 2 flacons de NAD/NADH recycling enzyme mixture au catalogue sous les références suivantes :

NAD/NADH Recycling Enzyme Mixture (ab189025)

NADH Sensor Buffer (ab189026)

Read More

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