NAD+/NADH Assay Kit (Colorimetric)

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme existing in oxidized (NAD⁺) and reduced (NADH) forms. As a central electron carrier, NAD⁺ participates in energy metabolism and regulates cellular processes through enzymatic reactions. For instance, Sirtuins deacetylases depend on NAD⁺ to modulate protein acetylation, thereby influencing gene expression and metabolism. NAD⁺ levels are also linked to apoptosis, redox balance, and signal transduction. Thus, the NAD⁺/NADH ratio serves as a key indicator of cellular energy status, redox homeostasis, and pathological mechanisms.

This kit uses a colorimetric method to measure NAD⁺ concentration, NADH concentration and NAD⁺/NADH ratio in the sample. In this assay, ethanol dehydrogenase (ADH) catalyzes the reaction of ethanol to acetaldehyde during the reduction of NAD⁺ to NADH. Subsequently, NADH reacts with WST-8 to produce an orange-yellow formazan with a maximum absorbance at 450nm. The amount of formazan is proportional to the amount of total NAD (NAD⁺+NADH). For the specific determination of NADH, heat samples at 60°C for 30 min to completely degrade NAD⁺, and perform the same reaction system to quantify the NADH concentration only. Finally, calculate the NAD⁺/NADH ratio based on the total NAD and NADH concentration.

Figure 1. Typical standard curve and assay data. (A) Measure the NAD⁺/NADH ratio, (B) NAD⁺ and NADH concentration in Hela, 293T and Jurkat cells, (C) NAD⁺ and NADH calibration standard curves, which are not interfered with by NADPH.