Nicotinamide adenine dinucleotide phosphate (NADP) is an important cofactor involved in reactions in the pentose phosphate pathway (PPP) that is one of the metabolic pathways in cells. NADP exists as an oxidized form NADP⁺ and a reduced form NADPH in cells. NADPH is important for not only biosynthesis of fatty acids and cholesterol but also generation of reduced glutathione (GSH). Quantitative determination of NADP⁺ / NADPH has applications in research pertaining to energy transformation and redox state of cells or tissue.

NADP⁺ / NADPH Assay Kit provides a simple, reproducible, and sensitive tool for measuring NADP⁺ / NADPH concentration in cell lysates, serum, plasma and other biological liquid samples. The assay is based on an enzymatic cycling reaction. NADP⁺ is reduced to NADPH by glucose-6-phosphate dehydrogenase during the oxidation of glucose-6-phosphate. Then NADPH reacts with a colorimetric probe that produces a colored product that can be measured at 450 nm. The intensity of the product color is proportional to the NADP⁺ and NADPH within a sample.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Tubes to prepare sample dilutions
• MilliQ water or other type of double distilled water (ddH₂O)
• 50 mL conical tubes

1. Add 50 µL of the diluted NADPH Standards or NADP+ / NADPH samples to the 96-well microtiter plate.
2. Initiate the reaction by adding 70 µL Working Solution to each well. Carefully, shake the plate for a few seconds to mix.
3. Incubate the plate for 1-4 hours at room temperature with gentle mixing in the dark.
4. Measure the absorbance at 450 nm using a plate reader.