Abstract
The FoF1 ATP synthase (ATPase) is one of the most important protein complexes in energy metabolism. The isolation of functional ATPase complexes is fundamental to address questions about its assembly, regulation, and functions. This protocol describes the purification of intact and active ATPase from the model cyanobacterium Synechocystis sp. PCC 6803. Basis for purification is a 3×FLAG tag fused to the beta subunit. The ATPase is enzymatically active and its purity is demonstrated using mass spectrometry, denaturing, and blue-native PAGE. For complete details on the use and execution of this protocol, please refer to Song et al. (2022).