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Amplite® Fluorimetric Total NADP and NADPH Assay Kit *Red Fluorescence*

NADPH dose response was measured with Amplite® Fluorimetric Total NADP and NADPH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU is Ex/EM = 540/590 nm.
NADPH dose response was measured with Amplite® Fluorimetric Total NADP and NADPH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU is Ex/EM = 540/590 nm.
NADPH dose response was measured with Amplite® Fluorimetric Total NADP and NADPH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU is Ex/EM = 540/590 nm.
NADP+/NADPH ratio under normoxia and hypoxia in shScr or shACC1 H1975 cells (n = 6). A ratio of NADP+/NADPH was calculated after measuring each molecule separately with the Amplite Fluorimetric NADP/NADPH Ratio Assay Kit from AAT Bioquest, Inc (Sunnyvale, CA). Protocol was conducted as the manufacturer suggested and all values were normalized to protein content, as measured by the Pierce BCA kit, on similarly plated and treated samples done in parallel. Source: Graph from <strong>ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via &alpha;-ketoglutarate</strong> by Melissa M. Keenan, et al., <em>PLOS,</em> Oct. 2015.
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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. In addition, this assay has very low background since it is run in the red visible range that significantly reduces the interference from biological samples. The assay has demonstrated high sensitivity and low interference with 570 nm excitation 590 nm emission.

Platform


Absorbance microplate reader

Absorbance576 &plusmn; 5 nm
Recommended plateClear bottom

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare NADP/NADPH working solution (50 µL) 
  2. Add NADPH standards or test samples (50 µL)
  3. Incubate at room temperature for 15 minutes – 2 hours
  4. 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

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.

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

NADPH standard

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.

BLBLTSTS
NS1NS1......
NS2NS2......
NS3NS3  
NS4NS4  
NS5NS5  
NS6NS6  
NS7NS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
NS1 - NS750 µLSerial Dilutions (0.003 to 3 µM)
BL50 µLPBS
TS50 µLtest sample
  1. 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).

  2. 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.

  3. Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.

  4. 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


View all 60 citations: Citation Explorer
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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 &amp; 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 &amp; 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)
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 &amp; 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
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
Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1&alpha; Signaling Pathway
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

References


View all 40 references: Citation Explorer
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Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range
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