Amplite® Fluorimetric NAD Assay Kit *Blue 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 | 420 nm |
Emission | 480 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare NAD standards or test samples (50 µL)
- Add 20 µL Quest Fluor™ NAD Probe
- Add 20 µL Assay Solution
- Incubate at RT for 10 - 20 minutes
- Add 15 µL Enhancer Solution
- Incubate at RT for 10 - 20 min
- Monitor Fluorescence at 420/480 nm
Important notes
Thaw each kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NAD standard solution (1 mM):
Add 500 µL of ddH2O into the vial of NAD standard (Component D) to make 1 mM NAD stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15280
Add 10 µL of NAD standard solution into 990 µL H2O or PBS buffer to generate 10 µM NAD standard solution (NS7). Then take the 10 µM NAD standard solution and perform 1:3 serial dilutions in H2O or PBS to get remaining serial dilutions of NAD standard (NS1 - NS6). Note: Diluted NAD standard solution is unstable, and should be used within 4 hours.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NAD standards and test samples in a black/solid bottom 96-well microplate. NS = NAD 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.
Well | Volume | Reagent |
NS1-NS7 | 50 µL | serial dilution (0.01 to 10 µM) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | sample |
- Prepare NAD standards (NS), blank controls (BL), and test samples (TS) 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.
- Add 20 µL Quest Fluor™ NAD Probe (Component A) solution into each well of NAD standard, blank control, and test samples, mix well. For 384-well plate, use 10 µL of Quest Fluor™ NAD Probe (Component A) solution instead.
- Add 20 µL Assay Solution (Component B) into each well, mix well. For 384-well plate, use 10 µL of Assay Solution (Component B) instead.
- Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.
- Add 15 µL Enhancer (Component C) to each well to make the total NAD assay volume of 105 µL/well, and incubate at room temperature for 10 - 20 minutes, protected from light. For a 384-well plate, add 7.5 uL Enhancer (Component C) instead, for a total volume of 52.5 µL/well.
- Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.
Images
Citations
Authors: Zou, Dianxiong and Ganugula, Raghu and Arora, Meenakshi and Nabity, Mary B and Sheikh-Hamad, David and Kumar, MNV Ravi
Journal: American Journal of Physiology-Renal Physiology (2019)
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: 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
Authors: Yang, Fei and Heit, Caitlin and Inglis, Debra L
Journal: Fermentation (2017): 61
Authors: Li, Miao and Liu, Xin and He, Yongpeng and Zheng, Qingyin and Wang, Min and Wu, Yu and Zhang, Yuanpeng and Wang, Chaoyun
Journal: European Journal of Pharmacology (2017): 124--133
Authors: Saihara, Kazuhiro and Kamikubo, Ryosuke and Ikemoto, Kazuto and Uchida, Koji and Akagawa, Mitsugu
Journal: Biochemistry (2017)
Authors: Wang, Chen and Cai, Heng and Chen, Zhongjun and Zhou, Zhihui
Journal: Biotechnology letters (2016): 1791--1797
Authors: Shao, Minglong and Zhang, Xian and Rao, Zhiming and Xu, Meijuan and Yang, Taowei and Li, Hui and Xu, Zhenghong and Yang, Shangtian
Journal: Green Chemistry (2016): 1774--1784
Application notes
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