Amplite™ Fluorimetric NADPH Assay Kit *Red Fluorescence*

Image Viewer
Analyze with Quest Graph™Plan a serial dilution
Close (X)
2e+41e+45.0e+380604020- NADP- NADPHData legend Generated with Quest Graph™ NADPH or NADP (uM) RFU Hover mouse to interact
NADPH dose response was measured with Amplite™ Fluorimetric NADPH Assay Kit in a 96-well solid black plate using a NOVOStar microplate reader (BMG Labtech). RFU measured over Ex/Em = 540/590 nm.
Loading...
 
Unit Size: Cat No: Price (USD): Qty:
400 Tests 15262 $295


Export item/cart as Excel file

Send item/cart as email
EXPORT TO EXCEL X

Export:
EXPORT TO EMAIL X
Important: We request your email address to ensure that the recipient(s) knows you intended for them to see the email, and that it is not junk mail.
Export:
Your Name*:
Your Email*:
Recipient Email*:
Your Personal Message:
Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
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™ NADPH Assay Kit provides a convenient method for sensitive detection of NADPH. The enzymes in the system specifically recognize NADPH in an enzyme cycling reaction. The enzyme cycling reaction significantly increases detection sensitivity.




Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare 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 increase at Ex/Em = 540/590 nm

Important notes
Thaw one of each kit component at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Recommended plate:Solid black
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.

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/15262

Use NADPH standard solution and PBS buffer (pH 7.4) to generate 100 µM (100 pmol/µL) NADPH standard solution (NS7). Then use 100 µM NADPH standard solution to perform 1:3 serial dilutions to get remaining serial dilutions of NADPH standard (NS6 - NS1). Note: Diluted NADPH standard solution is unstable, and should be used within 4 hours.

Preparation of working solution

Add 10 mL of Amplite™ NADPH Assay Buffer (Component B) into the bottle of NADPH Recycling Enzyme Mixture (Component A); mix well. Note: This 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 standard (NS1 - NS7, 0.1 to 100 µM); BL = blank control; TS = test sample.

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 Dilution (0.1 to 100 µM)
BL 50 µL PBS
TS 50 µL Test Sample
  1. Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Prepare cells or tissue samples as desired.

  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, use 25 µL of 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 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to fluorescence reading. For cell based NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate NADPH or NADP samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. NADPH dose response was measured with Amplite™ Fluorimetric NADPH Assay Kit in a 96-well solid black plate using a NOVOStar microplate reader (BMG Labtech). RFU measured over Ex/Em = 540/590 nm.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Celastrol attenuates angiotensin II mediated human umbilical vein endothelial cells damage through activation of Nrf2/ERK1/2/Nox2 signal pathway
Authors: Miao Li, Xin Liu, Yongpeng He, Qingyin Zheng, Min Wang, Yu Wu, Yuanpeng Zhang, Chaoyun Wang
Journal: European Journal of Pharmacology (2017): 124--133

Cytosolic Redox Status of Wine Yeast (Saccharomyces Cerevisiae) under Hyperosmotic Stress during Icewine Fermentation
Authors: Fei Yang, Caitlin Heit, Debra L Inglis
Journal: Fermentation (2017): 61

Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase
Authors: Min Ling, Peixin Huang, Shamima Islam, Daniel P Heruth, Xuanan Li, Li Qin Zhang, Ding-You Li, Zhaohui Hu, Shui Qing Ye
Journal: Cell & Bioscience (2017): 27

MCU-dependent mitochondrial Ca2+ inhibits NAD+/SIRT3/SOD2 pathway to promote ROS production and metastasis of HCC cells
Authors: T Ren, H Zhang, J Wang, J Zhu, M Jin, Y Wu, X Guo, L Ji, Q Huang, H Yang
Journal: Oncogene (2017)

Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase
Authors: Li Q Zhang, Leon Van Haandel, Min Xiong, Peixin Huang, Daniel P Heruth, Charlie Bi, Roger Gaedigk, Xun Jiang, Ding-You Li, Gerald Wyckoff
Journal: Cell Death & Disease (2017): e2705

Pyrroloquinoline Quinone, a Redox-active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating SIRT1/PGC-1α Signaling Pathway
Authors: Kazuhiro Saihara, Ryosuke Kamikubo, Kazuto Ikemoto, Koji Uchida, Mitsugu Akagawa
Journal: Biochemistry (2017)

Resveratrol attenuates excessive ethanol exposure induced insulin resistance in rats via improving NAD+/NADH ratio
Authors: Gang Luo, Bingqing Huang, Xiang Qiu, Lin Xiao, Ning Wang, Qin Gao, Wei Yang, Liping Hao
Journal: Molecular Nutrition & Food Research (2017)

A Snapshot of the Plant Glycated Proteome STRUCTURAL, FUNCTIONAL, AND MECHANISTIC ASPECTS
Authors: Tatiana Bilova, Elena Lukasheva, Dominic Brauch, Uta Greifenhagen, Gagan Paudel, Elena Tarakhovskaya, Nadezhda Frolova, Juliane Mittasch, Gerd Ulrich Balcke, Alain Tissier
Journal: Journal of Biological Chemistry (2016): 7621--7636

AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD+ elevation
Authors: Xiaojuan Han, Haoran Tai, Xiaobo Wang, Zhe Wang, Jiao Zhou, Xiawei Wei, Yi Ding, Hui Gong, Chunfen Mo, Jie Zhang
Journal: Aging cell (2016): 416--427

Cell-Line Selectivity Improves the Predictive Power of Pharmacogenomic Analyses and Helps Identify NADPH as Biomarker for Ferroptosis Sensitivity
Authors: Kenichi Shimada, Miki Hayano, Nen C Pagano, Brent R Stockwell
Journal: Cell chemical biology (2016): 225--235


View More Citations