Amplite® Colorimetric NADP/NADPH Ratio Assay Kit
Price | |
Catalog Number | |
Unit Size | |
Quantity |
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 25 µL of NADPH standards and/or test samples
- Add 25 µL of NADP Extraction Solution
- Incubate at 37 °C for 15 minutes
- Add 25 µL of Neutralization Solution
- Add 75 µL of NADP/NADPH working solution
- Incubate at RT for 15 minutes to 2 hours
- Monitor Absorbance at 460 nm
- It is highly recommended to incubate the cells with Lysis Buffer (Component G) at 37 °C 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 have 1 mM (1 nmol/µL) NADPH stock solution.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/15274
PREPARATION OF WORKING SOLUTION
- Add 8 mL of NADPH Probe buffer (Component B-II) to the bottle of NADP/NADPH Recycling Enzyme Mixture (Component A), and mix well.
- Add 2 mL NADPH Probe (Component B-I) into the same bottle (from Step 1) and mix well.
Note This NADP/NADPH working solution is enough for 125-200 assays. 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 white/clear bottom 96-well microplate. NS= NADPH Standards (NS1-NS7, 0.156 to 10 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.
Note High concentration of NADPH (e.g., >30 µM, final concentration) will cause saturated signal and make the calibration curve non-linear.
Well | Volume | Reagent |
NS1 - NS7 | 50 µL | Serial Dilutions (0.156 to 10 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.
Note Prepare cells or tissue samples as desired. Lysis Buffer (Component G) can be used for lysing the cells for convenience.
Note It is highly recommended to incubate the cells with Lysis Buffer (Component G) at 37oC and use the supernatant for the experiment. - Add 50 µL of NADP/NADPH working solution into each well of NADPH standard, blank control, and test samples to make the total NADP/NADPH assay volume of 100 µL/well.
- Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light. (We used 1 hour incubation time in data shown)
- Monitor the absorbance increase with an absorbance plate reader at 460 nm.
Table 3. Layout of NADPH standards and test samples in a white/clear 96-well microplate. NS= NADP/NADPH Standards (NS1 - NS7, 0.156 to 10 µM); BL=Blank Control; TS=Test Samples; TS (NADP) = Test Samples treated with NADP Extraction Solution (Component D) for 15 minutes, then neutralized by Neutralization Solution (Component E).
BL | BL | TS | TS | TS (NADP) | TS (NADP) |
NS1 | NS1 | ... | ... | ||
NS2 | NS2 | ... | ... | ||
NS3 | NS3 | ||||
NS4 | NS4 | ||||
NS5 | NS5 | ||||
NS6 | NS6 | ||||
NS7 | NS7 |
Table 4. Reagent compositions for each well.
Note High concentration of NADPH (e.g., >30 µM, final concentration) will cause saturated signal and make the calibration curve non-linear.
NADPH Standard | Blank Control | Test Sample (NADP+NADPH) | Test Sample (NADP Extract) |
Serial Dilutions: 25 µL | PBS: 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 |
Incubate at 37oC for 15 minutes | Incubate at 37oC for 15 minutes | Incubate at 37oC for 15 minutes | Incubate at 37oC for 15 minutes |
Component F: 25 µL | Component F: 25 µL | Component F: 25 µL | Component E: 25 µL |
Total: 75 µL | Total: 75 µL | Total: 75 µL | Total: 75 µL |
- Refer to Tables 3 & 4 for compositions of each well.
- For NADP Extraction (NADP amount): Add 25 µL of NADP 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 Neutralization Solution (Component E) to neutralize the NADP extracts as described in Tables 3 & 4.
- For Total NADP and NADPH (Total amount): Add 25 µL of NADP/NADPH Control Solution (Component F) into the wells of NADPH standards and NADP/NADPH containing test samples. Incubate at 37o C for 10 to 15 minutes, and then add 25 µL of Extraction Control Solution (Component F) as described in Tables 3 and 4.
Note Prepare cells or tissue samples as desired. Lysis Buffer (Component G) can be used for lysing the cells for convenience.
- Add 75 µL of NADP/NADPH working solution into each well of NADPH standard, blank control, and test samples (NADP/NADPH), and test sample (NADP Extract) to make the total assay volume of 150 µL/well.
- Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at 460 nm.
Images
Citations
Authors: Tong, Qingchao and Ma, Yilei and Gao, Yuzhen and Zhou, Lin and Fang, Qiang and Fang, Sining and Zhao, Ru and Zeng, Jin and Li, Guoli and Xie, Xinyou and others,
Journal: (2024)
Authors: Wang, Herong and Jiang, Guozhen and Liang, Nan and Dong, Tianyu and Shan, Mengying and Yao, Mingdong and Wang, Ying and Xiao, Wenhai and Yuan, Yingjin
Journal: Journal of Agricultural and Food Chemistry (2023)
Authors: Allie, Robert
Journal: (2022)
Authors: Khou, Sokchea and Popa, Alexandra and Luci, Carmelo and Bihl, Franck and Meghraoui-Kheddar, Aida and Bourdely, Pierre and Salavagione, Emie and Cosson, Estelle and Rubod, Alain and Cazareth, Julie and others,
Journal: Cancers (2020): 1860
Authors: Hu, Yanru and Xu, Wenzhao and Hu, Shishan and Lian, Lingdan and Zhu, Jing and Ren, Ang and Shi, Liang and Zhao, Ming Wen
Journal: Free Radical Biology and Medicine (2019)
Authors: Wu, Shourong and Wang, Huimin and Li, Yanjun and Xie, Yudan and Huang, Can and Zhao, Hezhao and Miyagishi, Makoto and Kasim, Vivi
Journal: Cancer research (2018): 4549--4562
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: Wiciarz, Monika and Niewiadomska, E and Kruk, Jerzy
Journal: Photosynthetica (2018): 811--819
Authors: Li, Liangzhi and Kang, Pei and Ju, Xin and Chen, Jiajia and Zou, Huibin and Hu, Cuiying and Yan, Lishi
Journal: Preparative Biochemistry and Biotechnology (2018): 257--263
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)
Application notes
Design of potent inhibitors of acetylcholinesterase using morin as the starting compound
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Induction of Neurite Outgrowth in PC12 Cells
Induction of Neuritogenesis in PC12 Cells by a Pulsed Electromagnetic Field
FAQ
Are NADH and ROS related?
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?
Will Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence* work with NADP/NADPH? Can this kit measure NADP+ and NADPH?