NAD is a complex organic molecule found in all living cells. It exists in two forms: oxidized (NAD+) and reduced (NADH). In cells, NAD+ acts as an electron acceptor, becoming reduced to NADH by accepting electrons from other molecules. In addition to electron transfer, NAD+ and NADH are also used as enzyme substrates to add or remove posttranslational modifications from proteins, such as in the process of ADP ribosylation. Quantitative determination of NAD+/NADH has applications in research related to energy transformation and redox state of cells or tissues.

NAD+/NADH Assay Kit provides a simple, reproducible, and sensitive tool of measuring NAD+/NADH concentration in cell lysates and other biological liquid samples. The assay is based on an enzymatic cycling reaction. NAD+ is reduced to NADH by alcohol dehydrogenase during the oxidation of ethanol to acetaldehyde. Then NADH reacts with a colorimetric probe that produces a colored product which can be measured at 450 nm. The intensity of the product color is proportional to the NAD+ and NADH 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
• Ultrapure water
• 15 mL conical tubes

1. Add 10 µL of the diluted NADH Standards or NAD+, NADH samples to the 96-well microtiter plate.
2. Initiate the reaction by adding 90 µL Working Solution to each well. Carefully shake the plate for a few seconds to mix.
3. Incubate the plate for 30 minutes at room temperature with gentle mixing in the dark.
4. Measure the absorbance at 450nm using a plate reader.