Amplite™ Fluorimetric NADP Assay Kit *Blue Fluorescence*

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


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200 Tests 15281 $345


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

Ex/Em (nm)422/466
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 for many enzyme reactions found in living cells. NAD 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 requires 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 used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. 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 NADPH or total NADP/NADPH amount, but detection NADP generation in the presence of large excess amount of NADPH has been quite challenging to date because NADP has its absorption peak at 260 nm and does not fluorescence, making the measurement unpractical. Amplite™ Fluorimetric NADP Assay Kit provides a sensitive and rapid detection of NADP. The kit directly measure NADP using Quest Fluor™ NADP reagent, our newly developed NADP sensor. The proprietary probe used in this kit reacts only with NADP to generate a product that fluorescence at Ex/Em = 420/480 nm, and has little response to NADPH. This kit can detect as little as 30 nM NADP in a 100 µL assay volume, and monitor 0.3% NADP generation in the presence of excess amount of NADPH. 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 NADP standards or test samples (50 µL)
  2. Add 20 µL Quest Fluor™ NADP 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 (Cutoff = 455 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
Cutoff:455 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. NADP standard solution (1 mM):
Add 500 µL of ddH2O into the vial of NADP Standard (Component D) to make 1 mM NADP standard solution.

Preparation of standard solution
NADP standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15281

Add 10 µL of 1mM NADP standard solution into 990 µL H2O or 1X PBS buffer to generate 10 µM NADP standard solution (NS7). Take 10 µM NADP standard solution (NS7) and perform 1:3 serial dilutions in H2O or 1X PBS buffer to get serially diluted NADP standard (NS6 - NS1). Note: Diluted NADP standard solution is unstable, and should be used within 4 hours.

Sample experimental protocol

Table 1. Layout of NADP standards and test samples in a black/solid bottom 96-well microplate. NS=NADP standards (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 1X PBS buffer 
TS 50 µL sample
  1. Prepare NADP standards (NS), blank controls (BL), and test samples (TS) 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.

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

  3. Add 20 µL Assay Solution (Component B) into each well, mix well. For a 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 NADP assay volume of 105 µL/well. For a 384-well plate, add 7.5 uL Enhancer (Component C) instead, for a total volume of 52.5 µL/well.

  6. Incubate at room temperature for 10 - 20 minutes, protected from light.

  7. Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm (Cutoff = 455nm).
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 NADP 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. NADP standard curve with 100 µM NADPH in presence in the solution. As low as 0.3% of NADP (~300 nM) converted from NADPH 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.





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Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase
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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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