Amplite® Fluorimetric NAD Assay Kit *Blue Fluorescence*

Protocol summary

1. Prepare NAD standards or test samples (50 µL)
2. Add 20 µL Quest Fluor™ NAD 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

Important notes
Thaw each kit components at room temperature before starting the experiment.

1. NAD standard solution (1 mM):
Add 500 µL of ddH₂O into the vial of NAD standard (Component D) to make 1 mM NAD stock solution.

Table 1. Layout of NAD standards and test samples in a black/solid bottom 96-well microplate. NS = NAD standard (NS1-NS7, 0.01 to 10 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare NAD standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 20 µL Quest Fluor™ NAD Probe (Component A) solution into each well of NAD standard, blank control, and test samples, mix well. For 384-well plate, use 10 µL of Quest Fluor™ NAD Probe (Component A) solution instead.
   
   
3. Add 20 µL Assay Solution (Component B) into each well, mix well. For 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 NAD assay volume of 105 µL/well, and incubate at room temperature for 10 - 20 minutes, protected from light. For a 384-well plate, add 7.5 uL Enhancer (Component C) instead, for a total volume of 52.5 µL/well.
   
   
6. Monitor the fluorescence increase with a fluorescence plate reader at 420/480 nm.