Amplite® Colorimetric NADH and NADPH Assay Kit
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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 | SDSProtocol |
Platform
Absorbance microplate reader
Absorbance | 460 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
- 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
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
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
https://www.aatbio.com/tools/serial-dilution/15272
PREPARATION OF 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.
Images
Citations
Authors: Qing, CY and Rujie, M and Yutong, L
Journal: J Gene Engg Bio Res (2023): 37--45
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
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
Authors: Allie, Robert
Journal: (2022)
Authors: Li, Jingquan and Zhang, Zihao and Feng, Xu and Shen, Zhuqing and Sun, Ji and Zhang, Xiuwen and Bu, Fengjiao and Xu, Midie and Tan, Cong and Wang, Ziliang
Journal: Cell Biology and Toxicology (2021): 1--14
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
Authors: Wang, Xiang and Balamurugan, Srinivasan and Liu, Si-Fen and Ji, Chang-Yang and Liu, Yu-Hong and Yang, Wei-Dong and Jiang, Liwen and Li, Hong-Ye
Journal: Journal of Experimental Botany (2021)
Authors: Wang, Xiang and Liu, Si-Fen and Qin, Zi-Hao and Balamurugan, Srinivasan and Li, Hong-Ye and Lin, Carol Sze Ki
Journal: Environmental Pollution (2020): 114854
Authors: Padder, Rayees Ahmad and Bhat, Zafar Iqbal and Zaki Ahmad, Neetu Singh and Husain, Mohammad
Journal: Frontiers in oncology (2020)
Authors: Wang, Xiang and Liu, Si-Fen and Li, Ruo-Yu and Yang, Wei-Dong and Liu, Jie-Sheng and Lin, Carol Sze Ki and Balamurugan, Srinivasan and Li, Hong-Ye
Journal: Biotechnology for biofuels (2020): 1--14
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FAQ
What assay kits measure NADP/NADPH from cell samples?
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