Amplite® Colorimetric Total NADP and NADPH Assay Kit

1. Prepare NADPH standards or test samples (50 µL)
2. Add NADP/NADPH working solution (50 µL)
3. Incubate at room temperature for 15 minutes – 2 hours
4. Monitor the absorbance intensity at 575 ± 5 nm

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 NADPH Standard (Component C) to make 1 mM (1 nmol/µL) NADPH standard solution.

Add 10 mL of NADPH Sensor Buffer (Component B) into the bottle of NADP/NADPH Recycling Enzyme Mix (Component A) and mix well to make NADP/NADPH working solution.

Note        This NADP/NADPH working solution is enough for two 96-well plates.

Table 1. Layout of NADPH standards and test samples in a white wall clear bottom 96-well microplate. NS= NADPH Standards (NS1 - NS7, 0.003 to 3 µM) , BL=Blank Control, TS=Test Samples.

Table 2. Reagent composition for each well. High concentration of NADPH (e.g., >100 µM, final concentration) may cause reduced signal due to the over oxidation of NADPH sensor.

1. Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   Note        Prepare cells or tissue samples as desired.
2. Add 50 µL of NADP/NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NADP/NADPH working solution into each well instead, for a total volume of 50 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 nm to ~605 nm to increase assay sensitivity.
   
   Note        For NADP/NADPH ratio measurements, kit 15263 is recommended. For cell based NADP/NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat #20012) is recommended to use for lysing the cells.