Amplite™ Fluorimetric NAD Assay Kit *Blue Fluorescence*

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1000100101- NAD in NADHData legend Generated with Quest Graph™ NAD Dose (µM in 100 µM in NADH) RFU Hover mouse to interact
NAD standard curve with 100 µM NADH in presence in the solution. As low as 0.3% of NAD (~300 nM) converted from NADH can be detected with 20 min incubation (n=3). RFU read at Ex/Em = 420/480 nm.


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200 Tests 15280 $345


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





Overview

Ex/Em (nm)422/466
Storage F/D/L
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
Nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD) are essential cofactors for many enzyme reactions found in living cells. Quantifying the generation or consumption of these factors is an important method to monitor the enzyme-mediated reaction or screening the modulator or substrate of these enzyme reactions. There are several kits on the market to quantify NADH or total NAD/NADH amount, but detection NAD generation in the presence of large excess amount of NADH has been quite challenging to date because NAD has its absorption peak at 259 nm and does not fluorescence, making the measurement unpractical. Amplite™ Fluorimetric NAD Assay Kit provides a sensitive and rapid detection of NAD. The kit directly measure NAD using Quest Fluor™ NAD reagent, our newly developed NAD sensor. The proprietary probe used in this kit reacts only with NAD to generate a product that fluorescence at Ex/Em = 420/480 nm, and has little response to NADH. This kit can detect as little as 30 nM NAD in a 100 µL assay volume, and monitor 0.3% NAD generation in the presence of excess amount of NADH. This assay can be performed in a convenient 96-well or 384-well microtiter-plate format and can be used in high-throughput screening.




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 NAD standards or test samples (50 µL)
  2. Add 20 µL Quest Fluor™ NAD Probe
  3. Add 20 µL Assay Solution
  4. Incubate at RT for 10 - 20 minutes
  5. Add 15 µL Enhancer Solution
  6. Incubate at RT for 10 - 20 min
  7. Monitor Fluorescence at 420/480 nm

Important notes
Thaw each kit components at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:420 nm
Emission:480 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. NAD standard solution (1 mM):
Add 500 µL of ddH2O into the vial of NAD standard (Component D) to make 1 mM NAD stock solution.

Preparation of standard solution
NAD standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15280

Add 10 µL of NAD standard solution into 990 µL H2O or PBS buffer to generate 10 µM NAD standard solution (NS7). Then take the 10 µM NAD standard solution and perform 1:3 serial dilutions in H2O or PBS to get remaining serial dilutions of NAD standard (NS1 - NS6). Note: Diluted NAD standard solution is unstable, and should be used within 4 hours.

Sample experimental protocol

Table 1. Layout of NAD standards and test samples in a black/solid bottom 96-well microplate. NS = NAD standard (NS1-NS7, 0.01 to 10 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 (0.01 to 10 µM)
BL 50 µL Assay Buffer
TS 50 µL sample
  1. Prepare NAD standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 20 µL Quest Fluor™ NAD Probe (Component A) solution into each well of NAD standard, blank control, and test samples, mix well. For 384-well plate, use 10 µL of Quest Fluor™ NAD Probe (Component A) solution instead.

  3. Add 20 µL Assay Solution (Component B) into each well, mix well. For 384-well plate, use 10 µL of Assay Solution (Component B) instead.

  4. Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.

  5. Add 15 µL Enhancer (Component C) to each well to make the total NAD assay volume of 105 µL/well, and incubate at room temperature for 10 - 20 minutes, protected from light. For a 384-well plate, add 7.5 uL Enhancer (Component C) instead, for a total volume of 52.5 µL/well.

  6. Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.
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 NAD Dose 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. NAD standard curve with 100 µM NADH in presence in the solution. As low as 0.3% of NAD (~300 nM) converted from NADH can be detected with 20 min incubation (n=3). RFU read at Ex/Em = 420/480 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


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