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Amplite® Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity*

NADH dose response was measured with the Amplite® Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity* in a 96-well white/clear bottom plate using a SpectraMax microplate reader (Molecular Devices).
NADH dose response was measured with the Amplite® Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity* in a 96-well white/clear bottom plate using a SpectraMax microplate reader (Molecular Devices).
NADH dose response was measured with the Amplite® Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity* in a 96-well white/clear bottom plate using a SpectraMax microplate reader (Molecular Devices).
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+, and NAD+ is the oxidized form of NADH. It forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is then used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. The traditional NAD/NADH and NADP/NADPH assays are done by monitoring of NADH or NADPH absorption at 340 nm. This method suffers low sensitivity and high interference since the assay is done in the UV range that requires expensive quartz microplate. This Amplite® NAD/NADH Assay Kit provides a convenient method for sensitive detection of 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 sample mix. The enzyme cycling reaction significantly increases detection sensitivity. Compared to Kit #15258, this kit has higher sensitivity.


Absorbance microplate reader

Absorbance460 nm
Recommended plateClear bottom


Example protocol


Protocol Summary
  1. Prepare NADH standards or test samples (50 µL)
  2. Add NAD/NADH working solution (50 µL)
  3. Incubate at RT for 15 minutes to 2 hours
  4. Monitor Absorbance at 460 nm 
Important      Thaw one of each kit component at room temperature before starting the experiment.


For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

NADH standard solution
Add 200 µL of PBS buffer into the vial of NADH standard (Component C) to make 1 mM (1 nmol/µL) NADH stock solution.


For convenience, use the Serial Dilution Planner:

NADH standard
Add 10 µL of 1 mM NADH stock solution into 990 µL PBS buffer (pH 7.4) to generate 10 µM (10 pmols/µL) NADH standard solution (NS7). Then take the 10 µM NADH standard solution and perform 1:2 serial dilutions to get remaining serially diluted NADH standards (NS1 - NS6). Note: Diluted NADH standard solution is unstable, and should be used within 4 hours.


  1. Add 8 mL of NADH Probe buffer (Component B-II) to the bottle of NAD/NADH Recycling Enzyme Mixture (Component A) and mix well.
  2. Add 2 mL of NADH Probe (Component B-I) into above bottle (from Step 1) and mix well.
    Note     This NAD/NADH working solution is enough for 200 assays. The working solution is not stable, use it promptly and avoid direct exposure to light. 


Table 1. Layout of NADH standards and test samples in a white wall clear bottom 96-well microplate. NS = NADH standard (NS1 - NS7, 0.156 to 10 µM); BL = blank control; TS = test sample.
Table 2. Reagent composition for each well. Note: High concentration of NADH (e.g., >100 µM, final concentration) will cause saturated signal and make the calibration curve non-linear.
NS1 - NS750 µLSerial Dilution (0.156 to 10 µM)
TS50 µLTest Sample
  1. Prepare NADH standards (NS), blank controls (BL), and test samples (TS) into a white wall clear bottom 96-well microplate according to the layout provided in Table 1 and Table 2. For 384-well plate, use 25 µL of reagent per well instead of 50 µL.
    Note     Prepare cells or tissue samples as desired. Lysis Buffer (Component D) can be used for lysing the cells for convenience.
  2. Add 50 µL of NAD/NADH working solution into each well of NADH standard, blank control, and test samples to make the total NADH/NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
  3. Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.
  4. Monitor the absorbance increase with an absorbance plate reader at 460 nm. 



View all 61 citations: Citation Explorer
NAD+ Synthetase is Required for Free-living and Symbiotic Nitrogen Fixation in the Actinobacterium Frankia casuarinae
Authors: Kucho, Ken-ichi and Asukai, Koya and Van Nguyen, Thanh
Journal: Microbes and Environments (2023): ME22093
NAD (H)-loaded nanoparticles for efficient sepsis therapy via modulating immune and vascular homeostasis
Authors: Ye, Mingzhou and Zhao, Yi and Wang, Yuyuan and Xie, Ruosen and Tong, Yao and Sauer, John-Demian and Gong, Shaoqin
Journal: Nature Nanotechnology (2022): 1--11
Enhanced 1, 3-propanediol production in Klebsiella pneumoniae by a combined strategy of strengthening the TCA cycle and weakening the glucose effect
Authors: Lu, Xinyao and Ren, Shunli and Lu, Jingzheng and Zong, Hong and Song, Jian and Zhuge, Bin
Journal: Journal of applied microbiology (2018)
Cytosolic Redox Status of Wine Yeast (Saccharomyces Cerevisiae) under Hyperosmotic Stress during Icewine Fermentation
Authors: Yang, Fei and Heit, Caitlin and Inglis, Debra L
Journal: Fermentation (2017): 61
Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1α Signaling Pathway
Authors: Saihara, Kazuhiro and Kamikubo, Ryosuke and Ikemoto, Kazuto and Uchida, Koji and Akagawa, Mitsugu
Journal: Biochemistry (2017)
Celastrol attenuates angiotensin II mediated human umbilical vein endothelial cells damage through activation of Nrf2/ERK1/2/Nox2 signal pathway
Authors: Li, Miao and Liu, Xin and He, Yongpeng and Zheng, Qingyin and Wang, Min and Wu, Yu and Zhang, Yuanpeng and Wang, Chaoyun
Journal: European Journal of Pharmacology (2017): 124--133
Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase
Authors: Zhang, Li Q and Van Ha, undefined and el, Leon and Xiong, Min and Huang, Peixin and Heruth, Daniel P and Bi, Charlie and Gaedigk, Roger and Jiang, Xun and Li, Ding-You and Wyckoff, Gerald and others, undefined
Journal: Cell Death & Disease (2017): e2705
MCU-dependent mitochondrial Ca2+ inhibits NAD+/SIRT3/SOD2 pathway to promote ROS production and metastasis of HCC cells
Authors: Ren, T and Zhang, H and Wang, J and Zhu, J and Jin, M and Wu, Y and Guo, X and Ji, L and Huang, Q and Yang, H and others, undefined
Journal: Oncogene (2017)
Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase
Authors: Ling, Min and Huang, Peixin and Islam, Shamima and Heruth, Daniel P and Li, Xuanan and Zhang, Li Qin and Li, Ding-You and Hu, Zhaohui and Ye, Shui Qing
Journal: Cell & Bioscience (2017): 27