Amplite® Fluorimetric Total NADP and 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
Absorbance microplate reader
Absorbance | 576 ± 5 nm |
Recommended plate | Clear bottom |
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 NADP/NADPH working solution (50 µL)
- Add NADPH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes – 2 hours
- Monitor fluorescence intensity 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/15259
Add 10 µL of 1 mM NADPH standard solution to 990 µL PBS buffer to generate 10 µM (10 pmol/µL) NADPH standard solution. Take the 10 µM NADPH standard solution to perform 1:3 serial dilutions to get serial dilutions of NADPH standard (NS7 - NS1). Note: Diluted NADPH standard solution is unstable and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 10 mL of NADP/NADPH Sensor Buffer (Component B) into the bottle of NADP/NADPH Recycling Enzyme Mixture (Component A) and mix well. Note: This NADP/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 Standards (NS1 - NS7, 0.003 to 3 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.
Well | Volume | Reagent |
NS1 - NS7 | 50 µL | Serial Dilutions (0.003 to 3 µM) |
BL | 50 µL | PBS |
TS | 50 µL | test sample |
- Prepare NADPH 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. High concentration of NADPH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADPH sensor (to a non-fluorescent product).
- 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, add 25 µL of NADPH 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, Emission = 590 - 600 nm (optimal at 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 NADP/NADPH ratio measurements, Cat No. 15264 is recommended. For cell based NADP/NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.
Images
Citations
Authors: Allie, Robert
Journal: (2022)
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: 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: Ali, Yousuf O and Allen, Hunter M and Yu, Lei and Li-Kroeger, David and Bakhshizadehmahmoudi, Dena and Hatcher, Asante and McCabe, Cristin and Xu, Jishu and Bjorklund, Nicole and Taglialatela, Giulio and others, undefined
Journal: PLoS Biol (2016): e1002472
References
Authors: Marino D, Gonzalez EM, Frendo P, Puppo A, Arrese-Igor C.
Journal: Planta. (2006)
Authors: Diaz-Flores M, Ibanez-Hern and ez MA, Galvan RE, Gutierrez M, Duran-Reyes G, Medina-Navarro R, Pascoe-Lira D, Ortega-Camarillo C, Vilar-Rojas C, Cruz M, Baiza-Gutman LA.
Journal: Life Sci (2006): 2601
Authors: Pedersen A, Johansson T, Rydstrom J, Goran Karlsson B.
Journal: Biochim Biophys Acta (2005): 254
Authors: Gaetani GF, Ferraris AM, Sanna P, Kirkman HN.
Journal: Biochem J (2005): 763
Authors: Bhatt AN, Shukla N, Aliverti A, Zanetti G, Bhakuni V.
Journal: Protein Sci (2005): 980
Authors: Barron JT, Sasse MF, Nair A.
Journal: Mol Cell Biochem (2004): 91
Authors: Ziegler GA, Schulz GE.
Journal: Biochemistry (2000): 10986
Authors: Peake SJ, Venning JD, Cotton NP, Jackson JB.
Journal: Biochim Biophys Acta (1999): 81
Authors: Minard KI, Jennings GT, Loftus TM, Xuan D, McAlister-Henn L.
Journal: J Biol Chem (1998): 31486
Authors: Ruiz C, Alegria A, Barbera R, Farre R, Lagarda MJ.
Journal: Eur J Clin Chem Clin Biochem (1997): 893
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