Amplite™ Colorimetric NAD/NADH Ratio Assay Kit

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Amplite™ Colorimetric NAD/NADH Ratio Assay Kit is used to measure total NAD/NADH amount and in a white/clear 96-well microplate using a SpectraMax microplate reader (Molecular devices).


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250 Tests 15273 $345


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Overview

Ex/Em (nm)460/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. 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. Our Amplite™ NAD/NADH Ratio Assay Kit provides a convenient method for sensitive detection of NAD, NADH and their ratio. The NADH probe is a chromogenic sensor that has its maximum absorbance at ~460 nm upon NADH reduction. The absorbance increase at ~460 nm is directly proportional to the concentration of NADH in the solution. The NADH probe can recognize NADH in an enzyme-free reaction, and the signal can be easily read by an absorbance microplate reader at ~460 nm. The Amplite™ Colorimetric NADH Assay Kit provides a sensitive assay to detect as little as 3 µM NADH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.




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 25 µL of NADH standards and/or test samples
  2. Add 25 µL of NAD Extraction Solution
  3. Incubate at room temperature for 15 minutes
  4. Add 25 µL of Neutralization Solution
  5. Add 75 µL of NAD/NADH working solution
  6. Incubate at RT for 15 minutes to 2 hours
  7. Monitor Absorbance at 460 nm

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

Key parameters
Instrument:Absorbance microplate reader
Absorbance:460 nm
Recommended plate:Clear bottom
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/15273

Add 10 µL of 1 mM (1 nmol/µL) NADH standard solution into 990 µL 1X PBS buffer (pH 7.4) to generate 10 µM (10 pmols/µL) NADH standard solution. Then take 10 µM NADH standard solution and perform 1:2 serial dilutions in 1X PBS buffer to get serially diluted NADH standards (NS7 - NS1). Note: Diluted NADH standard solution is unstable, and should be used within 4 hours. 

Preparation of working solution

1. Add 8 mL of NADH Probe Buffer (Component B-II) to the bottle of NAD/NADH Recycling Enzyme Mix (Component A), and mix well.

2. Add 2 mL NADH Probe (Component B-I) into the bottle of Component A+B-II and mix well to make NAD/NADH working solution. Note: This NAD/NADH working solution is enough for 125-200 assays. The working solution is not stable, use it promptly and avoid direct exposure to light.

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

Sample experimental protocol

TOTAL NAD/NADH Assay (avail. 400 assays/kit):

Table 1. Layout of NADH standards and test samples in a white/clear bottom 96-well microplate. NS= NADH Standards (NS1 - NS7, 0.078 to 5 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) will cause saturated signal and make the calibration curve non-linear.

Well Volume Reagent
NS1 - NS7 50 µL Serial Dilutions (0.078 to 5 µM)
BL 50 µL 1X PBS
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. Note: Prepare cells or tissue samples as desired. Lysis Buffer (Component G) can be used for lysing the cells for convenience.

  2. Add 50 µL of NAD/NADH working solution into each well of NADH standard, blank control, and test samples to make the total NAD/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 460 nm.

NAD/NADH RATIO Assay (avail. 250 assays/kit):

Table 3. Layout of NADH standards and test samples in a white/clear 96-well microplate. NS= NAD/NADH Standards (NS1 - NS7, 0.078 to 5 µM); BL=Blank Control; TS=Test Samples; TS (NAD) = Test Samples treated with NAD Extraction Solution (Component D) for 10 to 15 minutes, then neutralized by Neutralization Solution (Component E).

BL BL TS TS TS (NAD) TS (NAD)
NS1 NS1 ... ...    
NS2 NS2 ... ...    
NS3 NS3        
NS4 NS4        
NS5 NS5        
NS6 NS6        
NS7 NS7        

Table 4. Reagent compositions for each well. High concentration of NADH (e.g., >100 µM, final concentration) will cause saturated signal and make the calibration curve non-linear.

NADPH Standard Blank Control Test Sample (NAD/NADH) Test Sample (NAD Extract)
Serial Dilutions: 25 µL 1X PBS: 25 µL Test Sample: 25 µL Test Sample: 25 µL
Component F: 25 µL Component F: 25 µL Component F: 25 µL Component D: 25 µL
Incubate at room temperature for 10 to 15 minutes
Component F: 25 µL Component F: 25 µL Component F: 25 µL Component E: 25 µL
Total: 75 µL Total: 75 µL Total: 75 µL Total: 75 µL
  1. Refer to Tables 3 & 4 for compositions of each well.

    1. For NADH Extraction (NADPH amount): Add 25 µL of NAD Extraction Solution (Component D) into the wells of NAD/NADH containing test samples. Incubate at room temperature for 10 to 15 minutes, then add 25 µL of Neutralization Solution (Component E) to neutralize the NAD extracts as described in Tables 3 & 4.

    2. For Total NAD and NADH (Total amount): Add 25 µL of Extraction Control Solution (Component F) into the wells of NADH standards and NAD/NADH containing test samples. Incubate at room temperature for 10 to 15 minutes, and then add 25 µL of Extraction Control Solution (Component F) as described in Tables 3 and 4. Note: Prepare cells or tissue samples as desired. Lysis Buffer (Component G) can be used for lysing the cells for convenience.

  2. Add 75 µL of NAD/NADH working solution into each well of NADH standard, blank control, and test samples (NAD/NADH), and test sample (NAD Extract) to make the total assay volume of 150 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 460 nm.
Example data analysis and figures

The reading (Absorbance) 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 Total NAD/NADH 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. Amplite™ Colorimetric NAD/NADH Ratio Assay Kit is used to measure total NAD/NADH amount and in a white/clear 96-well microplate using a SpectraMax microplate reader (Molecular devices).

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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