AAT Bioquest

Amplite® Fluorimetric NAD Assay Kit *Blue Fluorescence*

Comparison of NAD and NADH response
Comparison of NAD and NADH response
Comparison of NAD and NADH response
NAD standard curve with 100 µM NADH in presence in the solution. As low as 0.3% of NAD (~300 nM) converted from NADH can be detected with 20 min incubation (n=3). RFU read at Ex/Em = 420/480 nm. 
Ordering information
Catalog Number
Unit Size
Add to cart
Additional ordering information
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD) are essential cofactors for many enzyme reactions found in living cells. Quantifying the generation or consumption of these factors is an important method to monitor the enzyme-mediated reaction or screening the modulator or substrate of these enzyme reactions. There are several kits on the market to quantify NADH or total NAD/NADH amount, but detection NAD generation in the presence of large excess amount of NADH has been quite challenging to date because NAD has its absorption peak at 259 nm and does not fluorescence, making the measurement unpractical. Amplite® Fluorimetric NAD Assay Kit provides a sensitive and rapid detection of NAD. The kit directly measure NAD using Quest Fluor™ NAD reagent, our newly developed NAD sensor. The proprietary probe used in this kit reacts only with NAD to generate a product that fluorescence at a specific excitation and emission spectra range, and has little response to NADH. This kit can detect as little as 30 nM NAD in a 100 µL assay volume, and monitor 0.3% NAD generation in the presence of excess amount of NADH. This assay can be performed in a convenient 96-well or 384-well microtiter-plate format and can be used in high-throughput screening.


Fluorescence microplate reader

Excitation420 nm
Emission480 nm
Recommended plateSolid black


Example protocol


Protocol Summary
  1. Prepare NAD standards or test samples (50 µL)
  2. Add 20 µL Quest Fluor™ NAD Probe
  3. Add 20 µL Assay Solution
  4. Incubate at RT for 10 - 20 minutes
  5. Add 15 µL Enhancer Solution
  6. Incubate at RT for 10 - 20 minutes
  7. Monitor Fluorescence at 420/480 nm

Important     Thaw each kit components at room temperature before starting the experiment.


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

NAD standard solution (1 mM)

Add 500 µL of ddH2O into the vial of NAD standard (Component D) to make 1 mM NAD stock solution.


For convenience, use the Serial Dilution Planner:

NAD standard
Add 10 µL of NAD standard solution into 990 µL ddH2O or 1X PBS buffer to generate 10 µM NAD standard solution (NS7). Then take the 10 µM NAD standard solution and perform 1:3 serial dilutions in ddH2O or PBS to get remaining serial dilutions of NAD standard (NS1 - NS6). Note: Diluted NAD standard solution is unstable, and should be used within 4 hours.


Table 1. Layout of NAD standards and test samples in a black/solid bottom 96-well microplate. NS = NAD standard (NS1-NS7, 0.01 to 10 µM); BL = blank control; TS = test sample.


Table 2. Reagent composition for each well.

NS1-NS750 µLserial dilution (0.01 to 10 µM)
BL50 µL

ddH2O or 1X PBS

TS50 µLsample
  1. Prepare NAD standards (NS), blank controls (BL), and test samples (TS) 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.
  2. Add 20 µL Quest Fluor™ NAD Probe (Component A) solution into each well of NAD standard, blank control, and test samples, mix well. For 384-well plate, use 10 µL of Quest Fluor™ NAD Probe (Component A) solution instead.
  3. Add 20 µL Assay Solution (Component B) into each well, mix well. For 384-well plate, use 10 µL of Assay Solution (Component B) instead.
  4. Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.
  5. Add 15 µL Enhancer (Component C) to each well to make the total NAD assay volume of 105 µL/well, and incubate at room temperature for 10 - 20 minutes, protected from light. For a 384-well plate, add 7.5 uL Enhancer (Component C) instead, for a total volume of 52.5 µL/well.
  6. Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.



View all 59 citations: Citation Explorer
Oral Delivery of Nanoparticle Urolithin A Normalizes Cellular Stress and Improves Survival in Mouse Model of Cisplatin-induced AKI
Authors: Zou, Dianxiong and Ganugula, Raghu and Arora, Meenakshi and Nabity, Mary B and Sheikh-Hamad, David and Kumar, MNV Ravi
Journal: American Journal of Physiology-Renal Physiology (2019)
Resveratrol attenuates excessive ethanol exposure induced insulin resistance in rats via improving NAD+/NADH ratio
Authors: Luo, Gang and Huang, Bingqing and Qiu, Xiang and Xiao, Lin and Wang, Ning and Gao, Qin and Yang, Wei and Hao, Liping
Journal: Molecular Nutrition & Food Research (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
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)
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
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
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
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)
AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD+ elevation
Authors: Han, Xiaojuan and Tai, Haoran and Wang, Xiaobo and Wang, Zhe and Zhou, Jiao and Wei, Xiawei and Ding, Yi and Gong, Hui and Mo, Chunfen and Zhang, Jie and others, undefined
Journal: Aging cell (2016): 416--427
Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)
Authors: Harlan, Benjamin A and Pehar, Mariana and Sharma, Deep R and Beeson, Gyda and Beeson, Craig C and Vargas, Marcelo R
Journal: Journal of Biological Chemistry (2016): 10836--10846