Amplite® Colorimetric Total NAD and NADH Assay Kit Enhanced Sensitivity

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.

Example protocol

AT A GLANCE

Protocol Summary

Prepare NADH standards or test samples (50 µL)
Add NAD/NADH working solution (50 µL)
Incubate at RT for 15 minutes to 2 hours
Monitor Absorbance at 460 nm

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

CELL PREPARATION

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

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 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.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15275

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.

PREPARATION OF WORKING SOLUTION

Add 8 mL of NADH Probe buffer (Component B-II) to the bottle of NAD/NADH Recycling Enzyme Mixture (Component A) and mix well.
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.

SAMPLE EXPERIMENTAL PROTOCOL

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.

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: High concentration of NADH (e.g., >100 µM, final concentration) will cause saturated signal and make the calibration curve non-linear.

Well	Volume	Reagent
NS1 - NS7	50 µL	Serial Dilution (0.156 to 10 µM)
BL	50 µL	PBS
TS	50 µL	Test Sample

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.
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.
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 460 nm.

Citations

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

The Linkage of Yeast Metabolites, Produced Under Hyperosmotic Stress, to Cellular Cofactor Systems During Icewine Fermentation.
Authors: Allie, Robert
Journal: (2022)

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

Page updated on December 17, 2024

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