Amplite™ Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*

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2e+42e+41e+46.0e+32.4e+31.8e+31.2e+3600- NADPH- NADHData legend Generated with Quest Graph™ NADH or NADPH (uM) RFU Hover mouse to interact
NADH dose response was measured with Amplite™ Total NAD and NADH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU at Ex/Em = 540/590 nm.


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400 Tests 15257 $295


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)571/585
Storage F/D/L
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™ NAD/NADH Assay Kit provides a convenient method for sensitive detection of NAD and NADH. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction. There is no need to purify NAD/NADH 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.








Protocol


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

Protocol summary

  1. Prepare NAD/NADH working solution (50 µL)
  2. Add NADH standards or test samples (50 µL)
  3. Incubate at room temperature for 15 minutes – 2 hours
  4. Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 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
Cutoff:570 nm
Recommended plate:Solid black
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. NADH standard solution (1 mM):
Add 200 µL of 1X PBS buffer into the vial of NADH Standard (Component C) to make 1 mM (1 nmol/µL) NADH standard solution.

Preparation of standard solution
NADH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15257

Add 10 µL of 1 mM (1 nmol/µL) NADH standard solution to 990 µL 1X PBS buffer to generate 10 µM (10 pmol/µL) NADH standard solution (NS7). Take 10 µM NADH standard solution (NS7) to perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted NADH standards (NS6 - NS1). Note: Diluted NADH standard solution is unstable and should be used within 4 hours.

Preparation of working solution

Add 10 mL of NADH Sensor Buffer (Component B) into the bottle of NAD/NADH Recycling Enzyme Mix (Component A) and mix well to make NAD/NADH working solution. Note: This NAD/NADH working solution is enough for two 96-well plates.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol

Table 1. Layout of NADH standards and test samples in a solid black bottom 96-well microplate. NS=NADH Standards (NS1 - NS7, 0.014 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. High concentration of NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product).

Well Volume Reagent
NS1 - NS7 50 µL Serial Dilutions (0.014 to 10 µM)
BL 50 µL 1X PBS buffer
TS 50 µL test sample
  1. Prepare NADH 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.

  2. Add 50 µL of NAD/NADH working solution to each well of NADH standard, blank control, and test samples to make the total NAD/NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NAD/NADH 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 Ex/Em = 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 NAD/NADH ratio measurements, kit 15263 is recommended. Note: For cell based NAD/NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat#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 NADH or NADPH 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. NADH dose response was measured with Amplite™ Total NAD and NADH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU at 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

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Authors: Fei Yang, Caitlin Heit, Debra L Inglis
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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


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