The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF OF 1) and its subcomplexes CF O and CF 1 were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569–575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF OF 1, CF O or CF 1 and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF 1 and ATP synthesis activity of reconstituted CF OF 1 were determined before and after dye removal. The secondary structure of CF O was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF OF 1 and CF 1 nor affects the subunit composition and the high α-helical content of CF O. In crystallization attempts, 2D arrays of CF OF 1 and of CF O before and after dye removal were obtained. In the aggregates of CF O, circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.