Amplite® Colorimetric Total NAD and NADH Assay Kit

1. Prepare NADH standards or test samples (50 µL)
2. Add NAD/NADH working solution (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     Thaw one of each kit component at room temperature before starting the experiment.

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 PBS buffer into the vial of NADH standard (Component C) to have 1 mM (1 nmol/µL) NADH stock 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. 

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.

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.

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.