Amplite® Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare NAD/NADH working solution (50 µL)
- Add NADH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes – 2 hours
- Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)
Important notes
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NADH standard solution (1 mM):
Add 200 µL of 1X PBS buffer into the vial of NADH Standard (Component C) to make 1 mM (1 nmol/µL) NADH standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15257
Add 10 µL of 1 mM (1 nmol/µL) NADH standard solution to 990 µL 1X PBS buffer to generate 10 µM (10 pmol/µL) NADH standard solution (NS7). Take 10 µM NADH standard solution (NS7) to perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted 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 NADH Sensor Buffer (Component B) into the bottle of NAD/NADH Recycling Enzyme Mix (Component A) and mix well to make NAD/NADH working solution. Note: This NAD/NADH working solution is enough for two 96-well plates.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NADH standards and test samples in a solid black bottom 96-well microplate. NS=NADH Standards (NS1 - NS7, 0.014 to 10 µM) , BL=Blank Control, TS=Test Samples.
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. High concentration of NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product).
Well | Volume | Reagent |
NS1 - NS7 | 50 µL | Serial Dilutions (0.014 to 10 µM) |
BL | 50 µL | 1X PBS buffer |
TS | 50 µL | test sample |
- Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Prepare cells or tissue samples as desired.
- Add 50 µL of NAD/NADH working solution to each well of NADH standard, blank control, and test samples to make the total NAD/NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NAD/NADH 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 fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm). Note:The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading. Note: For NAD/NADH ratio measurements, kit 15263 is recommended. Note: For cell based NAD/NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat#20012) is recommended to use for lysing the cells.
Images


Citations
Authors: Nishida, Takuto and Naguro, Isao and Ichijo, Hidenori
Journal: Cell Death Discovery (2022): 1--11
Authors: Allie, Robert
Journal: (2022)
Authors: Gan, Huoqun and Shen, Tian and Chupp, Daniel P and Taylor, Julia R and Sanchez, Helia N and Li, Xin and Xu, Zhenming and Zan, Hong and Casali, Paolo
Journal: Science advances (2020): eaay2793
Authors: Lu, Xinyao and Ren, Shunli and Lu, Jingzheng and Zong, Hong and Song, Jian and Zhuge, Bin
Journal: Journal of applied microbiology (2018)
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug design, development and therapy (2017): 3343
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)
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
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)
Authors: Li, J and Huang, Q and Long, X and Guo, X and Sun, X and Jin, X and Li, Z and Ren, T and Yuan, P and Huang, X and others,
Journal: Oncogene (2017): 4901--4912
Authors: Aitken, Laura and Baillie, Gemma and Pannifer, Andrew and Morrison, Angus and Jones, Philip S and Smith, Terry K and McElroy, Stuart P and Gunn-Moore, Frank J
Journal: SLAS DISCOVERY: Advancing Life Sciences R\&D (2017): 676--685
Application notes
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