Amplite® Fluorimetric NADPH 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 | SDSProtocol |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare NADPH working solution (50 µL)
- Add NADPH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes - 2 hours
- Monitor fluorescence increase at Ex/Em = 540/590 nm
Important notes
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NADPH standard solution (1 mM):
Add 200 µL of PBS buffer into the vial of NADPH standard (Component C) to make 1 mM (1 nmol/µL) NADPH stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15262
Use NADPH standard solution and PBS buffer (pH 7.4) to generate 100 µM (100 pmol/µL) NADPH standard solution (NS7). Then use 100 µM NADPH standard solution to perform 1:3 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
Add 10 mL of Amplite™ NADPH Assay Buffer (Component B) into the bottle of NADPH Recycling Enzyme Mixture (Component A); mix well. Note: This NADPH working solution is enough for two 96-well plates. The working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NADPH standards and test samples in a solid black 96-well microplate. NS = NADPH standard (NS1 - NS7, 0.1 to 100 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 (0.1 to 100 µM) |
BL | 50 µL | PBS |
TS | 50 µL | Test Sample |
- Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Prepare cells or tissue samples as desired.
- Add 50 µL of NADPH working solution to 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, use 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 fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to fluorescence reading. For cell based NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.
Images
Citations
Authors: Ezeri{\c{n}}a, Daria and Vo, Trung Nghia and Luo, Ting and Elkrim, Yvon and Suarez, Anna Escoda and Herinckx, Ga{\"e}tan and Vertommen, Didier and Laoui, Damya and Van Ginderachter, Jo A and Messens, Joris
Journal: Advances in Redox Research (2023): 100083
Authors: Allie, Robert
Journal: (2022)
Authors: Bera, Debbethi and Pal, Kunal and Ruidas, Bhuban and Mondal, Dheeraj and Pal, Shinjini and Paul, Biplab Kumar and Karmakar, Parimal and Das, Sukhen and Nandy, Papiya
Journal: Materials Today Communications (2020): 101099
Authors: Das, Sanghita and Bera, Debbethi and Pal, Kunal and Mondal, Dheeraj and Karmakar, Parimal and Das, Sukhen and Dey, Anindita
Journal: Journal of Drug Delivery Science and Technology (2020): 101994
Authors: Wang, Chaoyun and Wan, Hongzhi and Wang, Qiaoyun and Sun, Hongliu and Sun, Yeying and Wang, Kexin and Zhang, Chunxiang
Journal: Oxidative Medicine and Cellular Longevity (2020)
Authors: Ruidas, Bhuban and Sur, Tapas Kumar and Pal, Kunal and Som Chaudhury, Sutapa and Prasad, Parash and Sinha, Koel and Sarkar, Prasanta Kumar and Das, Pritha and Das Mukhopadhyay, Chitrangada
Journal: Molecular biology reports (2020): 3745--3763
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
References
Authors: Wang X, Shi L, Deng Y, Qu M, Mao S, Xu L, Xu W, Fang C.
Journal: Eur J Pharmacol (2015): 116
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