Amplite® Colorimetric NADH and NADPH Assay Kit

Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+. NAD forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. The traditional NAD/NADH and NADP/NADPH assays are based on monitoring the changes in NADH or NADPH absorption at 340 nm. The short UV wavelength of NAD/NADH and NADP/NADPH assays makes the traditional methods suffer low sensitivity and high interference. Due to the weak absorption of NAD and NADH, the UV absorption method requires large sample sizes, making NAD and NADH measurements unpractical for limited sample size. AAT Bioquest's Amplite® Colorimetric NADH and NADPH Assay Kit provides a convenient method for the detection of NADPH. The NADPH probe is a chromogenic sensor that has its maximum absorbance at ~460 nm upon NADH reduction. The absorbance increase at ~460 nm is directly proportional to the concentration of NADPH in the solution. The NADPH probe can recognize NADPH in an enzyme-free reaction, and the signal can be easily read by an absorbance microplate reader at ~ 460 nm. The Amplite® Colorimetric NADH and NADPH Assay Kit provides a sensitive assay to detect as little as 3 µM NADPH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Example protocol

AT A GLANCE

Protocol Summary

Prepare NADPH working solution (50 µL)
Add NADPH standards or test samples (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

NADPH standard solution (1 mM)

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

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15272

NADPH standard

Add 100 µL of NADPH standard solution into 400 µL PBS buffer (pH 7.4) to generate 200 µM (200 pmol/µL) NADPH standard solution (NS7). Take 200 µL of 200 µM NADPH standard solution to perform 1:2 serial dilutions to get remaining serial dilutions of NADPH standard (NS6 - NS1). Note: Diluted NADPH standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

NADH/NADPH Probe Working Solution

Add 1 mL of NADH/NADPH Probe (Component A) to 4 mL of NADPH Assay Buffer (Component B) and mix well.

Note: 5 mL of NADPH working solution is enough for one 96-well plate. The working solution is not stable. It should be used promptly and avoid direct exposure to light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of NADPH standards and test samples in a white/clear bottom 96-well microplate. NS = NADPH standard (NS1 - NS7, 3.13 to 200 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

Well	Volume	Reagent
NS1 - NS7	50 µL	serial dilution (3.13 to 200 µM)
BL	50 µL	PBS
TS	50 µL	sample

Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) into a white wall clear bottom 96-well microplate according to Table 1 and Table 2. For a 384-well plate, add 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 the NADH/NADPH probe working solution into each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of the NADH/NADPH probe 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; protect from light.
Monitor the absorbance increase with an absorbance plate reader at 460 nm.

Citations

View all 73 citations: Citation Explorer

Quantitative Risk Assessment of Product Disulfide Bond Reduction in A Recom-binant Protein Manufacturing
Authors: Qing, CY and Rujie, M and Yutong, L
Journal: J Gene Engg Bio Res (2023): 37--45

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

The Fibrillin-1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells
Authors: Wang, Ziliang and Chen, Wei and Zuo, Ling and Xu, Midie and Wu, Yong and Huang, Jiami and Zhang, Xu and Li, Yongheng and Wang, Jing and Chen, Jing and others,
Journal: Cancer Communications (2022): 245--265

PI3K-AKT Pathway Modulation by Thymoquinone Limits Tumor Growth and Glycolytic Metabolism in Colorectal Cancer
Authors: Karim, Shahid and Burzangi, Abdulhadi S and Ahmad, Aftab and Siddiqui, Nasir Ali and Ibrahim, Ibrahim M and Sharma, Priyanka and Abualsunun, Walaa A and Gabr, Gamal A
Journal: International Journal of Molecular Sciences (2022): 2305

Long non-coding RNA CTSLP8 mediates ovarian cancer progression and chemotherapy resistance by modulating cellular glycolysis and regulating c-Myc expression through PKM2
Authors: Li, Xiaoduan and Zhang, Yi and Wang, Xinjing and Lin, Feikai and Cheng, Xi and Wang, Ziliang and Wang, Xipeng
Journal: Cell Biology and Toxicology (2021): 1--19

Page updated on November 24, 2024

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