The NAD kinase activity assay is a pivotal biochemical technique used to measure the activity of NAD kinase, an enzyme responsible for phosphorylating NAD+ to produce NADP+. This conversion is crucial for maintaining the balance of the NAD/NADP+ pool, which plays a significant role in various metabolic processes, including cellular respiration, lipid synthesis, and the regulation of oxidative stress. The assay typically involves incubating a reaction mixture containing NAD+, ATP, and the enzyme source under controlled conditions. Following incubation, the reaction is terminated, and the resulting NADP+ can be quantified using spectrophotometric methods or high-performance liquid chromatography (HPLC).One common approach to this assay is to couple the reaction with glucose-6-phosphate dehydrogenase (G6PDH), where the production of NADP+ leads to the reduction of NAD+ to NADH. The increase in NADH can be measured at 340 nm, providing a direct assessment of NAD kinase activity. This assay is particularly valuable in research focused on metabolic regulation, cancer biology, and studying the effects of various compounds on cellular metabolism. Additionally, understanding NAD kinase activity has implications for therapeutic strategies targeting metabolic disorders and age-related diseases, making it an essential tool in both basic and applied research.