Amplite® Colorimetric NAD/NADH Ratio Assay Kit

1. Prepare 25 µL of NADH standards and/or test samples
2. Add 25 µL of NAD Extraction Solution
3. Incubate at 37^oC 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

It is highly recommended to incubate the cells with Lysis Buffer (Component G) at 37 °C and use the supernatant for the experiment.

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

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

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

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.

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

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.

Note         One can try to use ddH2O such as 500 uL to dissolve the Component A, and then mix with B-II and B-I proportionally to make the NAD/NADH working solution for enough use only.

Table 1. Layout of NADH standards and test samples in a white/clear bottom 96-well microplate. NS= NADH Standards (NS1 - NS7, 100 to 1.56 µM), BL=Blank Control, TS=Test Samples.

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.

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 and incubate the cells with Lysis Buffer at 37^oC for 15 minutes and use the supernatant for the experiment.
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.

Table 3. Layout of NADH standards and test samples in a white/clear 96-well microplate. NS= NADH Standards (NS1 - NS7, 100 to 1.56 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).

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.

1. Refer to Tables 3 & 4 for compositions of each well.

   1. For NAD Extraction (NAD amount): Add 25 µL of NAD Extraction Solution (Component D) into the wells of NAD/NADH containing test samples. Incubate at 37^oC 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 37^oC 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 (We tested 60 minutes in the figure shown), protected from light

4. Monitor the absorbance increase with an absorbance plate reader at 460 nm.