Amplite® Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*
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Additional ordering information
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 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Alternative formats
Amplite® Colorimetric Total NADP and NADPH Assay Kit |
Overview | SDSProtocol |
See also: Amplite® Reagents and Assay Kits, Cell Metabolism, Dehydrogenases, NAD/NADH and NADP/NADPH
Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+, and NAD+ is the oxidized form of NADH. It forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is then used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. The traditional NAD/NADH and NADP/NADPH assays are done by monitoring of NADH or NADPH absorption at 340 nm. This method suffers low sensitivity and high interference since the assay is done in the UV range that requires expensive quartz microplate. This Amplite® NADP/NADPH Assay Kit provides a convenient method for sensitive detection of NADP and NADPH. The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction. There is no need to purify NADP/NADPH from sample mix. The enzyme cycling reaction significantly increases detection sensitivity. Compared to Kit #15260, this kit has higher sensitivity.
Platform
Absorbance microplate reader
Absorbance | 460 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol Summary
- Prepare NADPH standards or test samples (50 µL)
- Add NADP/NADPH working solution (50 µL)
- Incubate at room temperature for 15 minutes to 2 hours
- Monitor Absorbance at 460 nm
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.
NADPH standard solution (1 mM)
Add 200 µL of 1X PBS buffer into the vial of NADPH Standard (Component C) to make 1 mM (1 nmol/µL) NADPH standard solution.PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15276
https://www.aatbio.com/tools/serial-dilution/15276
NADPH standard
Add 2 µL of 1 mM NADPH standard solution into 998 µL 1X PBS buffer (pH 7.4) to generate 2 µM (2 pmols/µL) NADPH standard solution (NS7). Take 2 µM NADPH standard solution (NS7) and perform 1:2 serial dilutions in 1X PBS buffer to get serially diluted NADPH standards (NS6 - NS1). Note: Diluted NADPH standard solution is unstable, and should be used within 4 hours.PREPARATION OF WORKING SOLUTION
- Add 8 mL of NADPH Probe Buffer (Component B-II) to the bottle of NADP/NADPH Recycling Enzyme Mix (Component A) and mix well.
- Add 2 mL of NADPH Probe (Component B-I) into the bottle of Component A+B-II and mix well to make NADP/NADPH working solution.
Note This NADP/NADPH working solution is enough for 200 assays.
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, 0.0313 to 2 µM), BL=Blank Control, TS=Test Samples.
Table 2. Reagent composition for each well. High concentration of NADPH (e.g., >30 µM, final concentration) will cause a saturated signal and make the calibration curve non-linear.
BL | BL | TS | TS |
NS1 | NS1 | ... | ... |
NS2 | NS2 | ... | ... |
NS3 | NS3 | ||
NS4 | NS4 | ||
NS5 | NS5 | ||
NS6 | NS6 | ||
NS7 | NS7 |
Well | Volume | Reagent |
NS1 - NS7 | 50 µL | Serial Dilutions (0.0313 to 2 µM) |
BL | 50 µL | 1X PBS buffer |
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. Lysis Buffer (Component D) can be used for lysing the cells for convenience. - Add 50 µL of NADP/NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADP/NADPH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NADP/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 absorbance increase with an absorbance plate reader at 460 nm.
Images
Citations
View all 59 citations: Citation Explorer
The Linkage of Yeast Metabolites, Produced Under Hyperosmotic Stress, to Cellular Cofactor Systems During Icewine Fermentation.
Authors: Allie, Robert
Journal: (2022)
Authors: Allie, Robert
Journal: (2022)
Enhanced 1, 3-propanediol production in Klebsiella pneumoniae by a combined strategy of strengthening the TCA cycle and weakening the glucose effect
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: Lu, Xinyao and Ren, Shunli and Lu, Jingzheng and Zong, Hong and Song, Jian and Zhuge, Bin
Journal: Journal of applied microbiology (2018)
Resveratrol attenuates excessive ethanol exposure induced insulin resistance in rats via improving NAD+/NADH ratio
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: 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)
Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase
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: 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
MCU-dependent mitochondrial Ca2+ inhibits NAD+/SIRT3/SOD2 pathway to promote ROS production and metastasis of HCC cells
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: 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)
Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase
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: 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
Cytosolic Redox Status of Wine Yeast (Saccharomyces Cerevisiae) under Hyperosmotic Stress during Icewine Fermentation
Authors: Yang, Fei and Heit, Caitlin and Inglis, Debra L
Journal: Fermentation (2017): 61
Authors: Yang, Fei and Heit, Caitlin and Inglis, Debra L
Journal: Fermentation (2017): 61
Celastrol attenuates angiotensin II mediated human umbilical vein endothelial cells damage through activation of Nrf2/ERK1/2/Nox2 signal pathway
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: 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
Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1α Signaling Pathway
Authors: Saihara, Kazuhiro and Kamikubo, Ryosuke and Ikemoto, Kazuto and Uchida, Koji and Akagawa, Mitsugu
Journal: Biochemistry (2017)
Authors: Saihara, Kazuhiro and Kamikubo, Ryosuke and Ikemoto, Kazuto and Uchida, Koji and Akagawa, Mitsugu
Journal: Biochemistry (2017)
Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O2 deprivation
Authors: Wang, Chen and Cai, Heng and Chen, Zhongjun and Zhou, Zhihui
Journal: Biotechnology letters (2016): 1791--1797
Authors: Wang, Chen and Cai, Heng and Chen, Zhongjun and Zhou, Zhihui
Journal: Biotechnology letters (2016): 1791--1797
Application notes
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Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity