N-His-PSBS purification by affinity chromatography. A, Immunoblotting with anti-His-tag antibody on different purification fractions obtained from affinity chromatography of N-His-PSBS line 2 thylakoids. Fractions corresponding to starting material (solubilized thylakoids), flow through, column washes, and eluate with 150 mm imidazole are shown. B, PSBS immunodetection (top) and SDS-PAGE profile (bottom) of solubilized thylakoids and eluate with 150 mm imidazole from N-His-PSBS and psbs KO samples. Arrows indicate the main profile differences in N-His-PSBS and psbs KO eluates. Red segments indicate the gel regions where the proteins identified in Table II should be localized. One microgram of Chl was loaded for solubilized thylakoids and flow through in both A and B. For chromatography eluates, a volume corresponding to 15 (immunoblotting) and 130 (SDS-PAGE) µg of Chl in starting thylakoids was loaded. C, Absorption spectra of solubilized thylakoids (black) and N-His-PSBS eluate (red). D, Absorption spectra of eluates from N-His-PSBS (red) and psbs KO (blue). Spectra were normalized to the maximum of the red absorption band. a.u., Arbitrary units.