Amplite® Fluorimetric NADP/NADPH Ratio 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
- Prepare 25 µL NADPH standards and/or test samples
- Add 25 µL of NADPH or NADP Extraction Solution
- Incubate at 37oC for 15 minutes
- Add 25 µL of NADP or NADPH Extraction Solution
- Add 75 µL NADP/NADPH working solution
Incubate at 37 °C for 15 minutes – 2 hours
- Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)
It is highly recommended to incubate the cells with Lysis Buffer (Component G) at 37oC and use the supernatant for the experiment.
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 into the vial of NADPH standard (Component C) to make 1 mM (1 nmol/µL) NADPH standard solution.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/15264
PREPARATION OF WORKING SOLUTION
Add 10 mL of NADPH Sensor Buffer (Component B) into the bottle of NADP/NADPH Recycling Enzyme Mixture (Component A) and mix well to make NADP/NADPH working solution.
Note This NADP/NADPH working solution is enough for 125 assays in 96-well plate.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NADPH standards and test samples in a solid black 96-well microplate. NS= NADPH Standards (NS1 - NS7, 0.01 to 10 µM); BL=Blank Control; TS=Test Samples; TS (NADPH) = Test Samples treated with NADPH Extraction Solution, then neutralized by NADP Extraction Solution; TS (NADP) = Test Samples treated with NADP Extraction Solution, then neutralized by NADPH Extraction Solution.
BL | BL | TS | TS | TS (NADPH) | TS (NADPH) | TS (NADP) | TS (NADP) |
NS1 | NS1 | ... | ... | ... | ... | ... | ... |
NS2 | NS2 | ... | ... | ... | ... | ... | ... |
NS3 | NS3 | ||||||
NS4 | NS4 | ||||||
NS5 | NS5 | ||||||
NS6 | NS6 | ||||||
NS7 | NS7 |
Table 2. Reagent composition for each well. Note: 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).
NADPH Standard | Blank Control | Test Sample (NADP+NADPH) | Test Sample (NADPH Extract) | Test Sample (NADP Extract) |
Serial Dilutions*: 25 μL | PBS: 25 μL | Test Sample: 25 μL | Test Sample: 25 μL | Test Sample: 25 μL |
Component F: 25 μL | Component F: 25 μL | Component F: 25 μL | Component D: 25 μL | Component E: 25 μL |
Incubate at 37 °C for 10 to 15 minutes | ||||
Component F: 25 μL | Component F: 25 μL | Component F: 25 μL | Component E: 25 μL | Component D: 25 μL |
Total: 75 μL | Total: 75 μL | Total: 75 μL | Total: 75 μL | Total: 75 μL |
- Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2.
Note Prepare cells or tissue samples as desired. NADP/NADPH Lysis Buffer (Component G) can be used for lysing the cells for convenience. One can simply use total NADP and NADPH minus the NADP to calculate the amount of NADPH.
Note It is highly recommended to incubate the cells at 37 °C and use the supernatant for the experiment. - For NADP Extraction (NADP): Add 25 µL of NADP Extraction Solution (Component E) into the wells of NADP/NADPH containing test samples. Incubate at 37 °C for 10 to 15 minutes, then add 25 µL of NADPH Extraction Solution (Component D) to neutralize the NADP extracts as described in Tables 1 & 2.For Total NADP and NADPH: Add 25 µL of NADP/NADPH Control Solution (Component F) into the wells of NADPH standards and NADP/NADPH containing test samples. Incubate at 37 °C for 10 to 15 minutes, and then add 25 µL of Control Solution (Component F) as described in Tables 1 & 2.For NADPH Extraction (NADPH): Add 25 µL of NADPH Extraction Solution (Component D) into the wells of NADP/NADPH containing test samples. Incubate at 37 °C for 10 to 15 minutes, then add 25 µL of NADP Extraction Solution (Component E) to neutralize the NADPH extracts as described in Tables 1 & 2.
- Add 75 µL of NADPH working solution into each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 150 µL/well.
- Incubate the reaction at 37 °C for 15 minutes to 2 hours (We tested 60 minutes in the figure shown), protected from light.
Note In some cases, incubation time can be increased to more than 2 hours. - Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 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.
Images
Citations
Authors: Yu, Wen and Zeng, Yue and Wang, Zenghao and Xia, Shengpeng and Yang, Zhiwen and Chen, Weijian and Huang, Yiming and Lv, Fengting and Bai, Haotian and Wang, Shu
Journal: Science Advances (2023): eadf6772
Authors: Allie, Robert
Journal: (2022)
Authors: Zhang, Ruihao and Zhu, Baohua and Tu, Changchao and Li, Yun and Zhao, Yan and Pan, Kehou
Journal: Journal of Applied Phycology (2021): 1--9
Authors: Zhou, Xin and Zeng, Yue and Tang, Yongyan and Huang, Yiming and Lv, Fengting and Liu, Libing and Wang, Shu
Journal: Science advances (2020): eabc5237
Authors: Luo, Hong-min and Wu, Xia and Xian, Xian and Wang, Lu-yao and Zhu, Liang-yu and Sun, Hong-yu and Yang, Lei and Liu, Wen-xuan
Journal: Journal of Cellular and Molecular Medicine (2020)
Authors: Zhu, Manlu and Dai, Xiongfeng
Journal: Nucleic acids research (2019)
Authors: Li, Haoxin and Ericsson, Maria and Rabasha, Bokang and Budnik, Bogdan and Chan, Sze Ham and Freinkman, Elizaveta and Lewis, Caroline A and Doench, John G and Wagner, Bridget K and Garraway, Levi A and others, undefined
Journal: Cell chemical biology (2019)
Authors: Han, Ting-Li and Cannon, Richard D and Gallo, S and ra M , undefined and Villas-Boas, Silas G
Journal: NPJ biofilms and microbiomes (2019): 13
Authors: Zhu, Bao-Hua and Zhang, Rui-Hao and Lv, Na-Na and Yang, Guan-Pin and Wang, Yi-Sheng and Pan, Ke-Hou
Journal: Frontiers in plant science (2018): 826
Authors: Liu, Yanyan and Cao, Yinglong and Zhang, Qinglu and Li, Xianghua and Wang, Shiping
Journal: Plant physiology (2018): 923--935
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FAQ
What assay kits measure NADP/NADPH from cell samples?
Why should I use an absorbance ratio at A575nm/A605nm when using most of your Amplite® Colorimetric Assay Kits?
How should I reconstitute an NADPH standard?
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