The mitochondrial F1FO ATP synthase, commonly referred to as complex V, is a multi-subunit protein that utilizes energy in the form of a proton gradient generated by the electron transport chain, to phosphorylate ADP to form ATP in the presence of Pi. This mechanism, referred to as oxidative phosphorylation, is responsible for ~90% of ATP generation in mammalian cells. Cayman's MitoCheck® Complex V Activity Assay measures the activity of Complex V, in the reverse reaction, as an ATPase in the mitochondrial samples provided. In this assay, ATP is dephosphorylated ADP by complex V. The ADP is then utilized by pyruvate kinase to convert phosphoenolpyruvate into pyruvate with the concomitant generation of ATP. Pyruvate, in the presence of NADH and lactate dehydrogenase, is then reduced to lactate and NAD+. The rate of NADH oxidation is monitored at 340 nm. Isolated bovine heart mitochondria are provided in this kit. Tissue mitochondria can also be acquired using Cayman’s MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).