Amplite® Colorimetric Total NAD and NADH Assay Kit

Name: Amplite® Colorimetric Total NAD and NADH Assay Kit
Brand: AAT Bioquest
SKU: 15258
Price: 341 USD
Availability: InStock

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.

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.

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)

Page updated on November 21, 2024

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