Amplite™ Colorimetric Total NAD and NADH Assay Kit

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0.360.270.189e-28642- NADPH- NADHData legend Generated with Quest Graph™ NADH or NADPH (uM) Absorbance (570 nm) Hover mouse to interact
NADH dose response was measured with Amplite™ Colorimetric Total NAD and NADH Assay Kit in a white/clear bottom 96-well plate using a NOVOStar (BMG Labtech) microplate reader.
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Unit Size: Cat No: Price (USD): Qty:
400 Tests 15258 $295


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Overview

Ex/Em (nm)575/None
Storage F/D/L
InstrumentsAbsorbance 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.




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 absorbance increase at the absorbance ratio of 570/610 nm.

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

Key parameters
Instrument:Absorbance microplate reader
Absorbance:570/610 nm
Recommended plate:Clear bottom
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. NADH stock solution (1 mM):
Add 200 µL of PBS buffer into the vial of NADH standard (Component C) to have 1 mM (1 nmol/µL) NADH stock solution.

Preparation of standard solution
NADH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15258

Add 10 µL of 1 mM NADH stock solution into 990 µL PBS buffer (pH 7.4) to generate 10 µM NADH standard solution (NS7). Then perform 1:3 serial dilutions to get serially diltued 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 NAD/NADH Sensor Buffer (Component B) to the bottle of NAD/NADH Recycling Enzyme Mixture (Component A), and mix well. Note: This NAD/NADH 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 NADH standards and test samples in a solid black 96-well microplate. NS = NADH 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. Note that high concentration of NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor.

Well Volume Reagent
NS1-NS7 50 µL serial dilution (0.01 to 10 µM)
BL 50 µL PBS
TS 50 µL sample
  1. Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout described in Tables 1 and 2. Prepare cells or tissue samples as desired. For a 384-well plate, use 25 µL of reagent per well isntead of 50 µL.

  2. Add 50 µL of NADH working solution into each well of NADH standard, blank control, and test samples to make the total NADH assay volume of 100 µL/well. 

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

  4. Monitor the absorbance increase with an absorbance plate reader at 575 ± 5 nm or at the absorbance ratio of 570/610 nm to increase assay sensitivity. Note: For NAD/NADH ratio measurements, Cat No. 15263 is recommended. For cell based NAD/NADH 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 (Absorbance (570 nm)) 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™ Colorimetric Total NAD and NADH Assay Kit in a white/clear bottom 96-well plate using a NOVOStar (BMG Labtech) microplate reader.

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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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

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Resveratrol attenuates excessive ethanol exposure induced insulin resistance in rats via improving NAD+/NADH ratio
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