Amplite® Colorimetric Total NAD and NADH Assay Kit

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Alternative formats

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

Overview SDS Protocol

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.

Platform

Absorbance microplate reader

Absorbance	570/610 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare NADH standards or test samples (50 µL)
Add NAD/NADH working solution (50 µL)
Incubate at room temperature for 15 minutes - 2 hours
Monitor absorbance increase at the absorbance ratio of 570/610 nm.

Important Thaw one of each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

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 stock solution (1 mM)

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

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15258

NADH standard

Add 10 µL of 1 mM NADH stock solution into 990 µL PBS buffer (pH 7.4) to generate 10 µM NADH standard solution (NS7). Then perform 1:3 serial dilutions to get serially diltued NADH standards (NS6 - NS1). Note: Diluted NADH standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 10 mL of NAD/NADH Sensor Buffer (Component B) to the bottle of NAD/NADH Recycling Enzyme Mixture (Component A), and mix well.
Note This NAD/NADH working solution is enough for two 96-well plates. The working solution is not stable, use it promptly and avoid direct exposure to light.

SAMPLE EXPERIMENTAL PROTOCOL

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

BL	BL	TS	TS
NS1	NS1	...	...
NS2	NS2	...	...
NS3	NS3
NS4	NS4
NS5	NS5
NS6	NS6
NS7	NS7

Table 2. Reagent composition for each well. Note that high concentration of NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor.

Well	Volume	Reagent
NS1-NS7	50 µL	serial dilution (0.01 to 10 µM)
BL	50 µL	PBS
TS	50 µL	sample

Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout described in Tables 1 and 2. Prepare cells or tissue samples as desired. For a 384-well plate, use 25 µL of reagent per well isntead of 50 µL.
Add 50 µL of NADH working solution into each well of NADH standard, blank control, and test samples to make the total NADH assay volume of 100 µL/well.
Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.
Monitor the absorbance increase with an absorbance plate reader at 575 ± 5 nm or at the absorbance ratio of 570/610 nm to increase assay sensitivity.
Note For NAD/NADH ratio measurements, Cat No. 15263 is recommended. For cell based NAD/NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.

Images

Figure 1. NADH dose response was measured with Amplite® Colorimetric Total NAD and NADH Assay Kit in a white/clear bottom 96-well plate using a NOVOStar (BMG Labtech) microplate reader.

Figure 2. PQQ increases in cellular NAD⁺ concentration. (A–D) NIH/3T3 fibroblasts were incubated with the indicated concentrations of PQQ for times ranging from 1 to 15 h (A and B) or for 6 h (C and D). After the incubation, the cellular levels of NAD⁺ (A and C) and total NAD⁺ and NADH (B and D) were measured and normalized to the cellular protein content. The results are shown as means ± SEM (n ≥ 3). N.S. means not significant. **p < 0.01 and ***p < 0.001 vs the vehicle-treated control (ANOVA, Dunnett’s multiple-comparison test). Source: Pyrroloquinoline Quinone, a Redox-Active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating the SIRT1/PGC-1α Signaling Pathway by Saihara et al., ACS Publications, Nov. 2017.

Citations

View all 64 citations: Citation Explorer

Nicotinamide ribose ameliorates myocardial ischemia/reperfusion injury by regulating autophagy and regulating oxidative stress
Authors: Yuan, Chen and Yang, Heng and Lan, Wanqi and Yang, Juesheng and Tang, Yanhua
Journal: Experimental and Therapeutic Medicine (2024): 1--10

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

Mycoplasma pneumoniae protects infected epithelial cells from hydrogen peroxide-induced cell detachment
Authors: Yamamoto, Takeshi and Kida, Yutaka and Kuwano, Koichi
Journal: Cellular microbiology (2019): e13015

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)

Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug design, development and therapy (2017): 3343

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