Enhanced chemiluminescence substrate detection kit

Total protein extracted from the fresh algal samples, after determined by a BCA Protein Assay Kit (Beyotime), was run with loading buffer (30 μg) on a 12% SDS-PAGE gel and subsequently transferred to a PVDF membrane, and immunoblotted with anti-PsbD (Agrisera, Sweden), anti-Cyt b6 (Agrisera), antiLhca2 (Agrisera) antibodies. Anti-Histone H3 (Abcam, USA) was selected as the internal reference. After the incubation with an anti-rabbit IgG antibody (BioXCell, USA), the antigen–antibody complexes on the membrane were visualized by using an enhanced chemiluminescence substrate detection kit (Thermo Fisher Scientific, USA) and captured on a ChemiDoc MP imaging system (Bio-Rad, USA).

Algal cells were disrupted using a mini bead-beater (Biospec, USA). The homogenates were centrifuged at 1,000 g for 3 min at 4°C to remove unbroken cells and cell debris. The supernatant was then transferred to a new tube and centrifuged at 12,000 g for 30 min at 4°C to obtain the crude membranes. The resulting pellets were resuspended in 60 µL SBA buffer containing 0.1 M dithiothreitol, 0.1 M Na₂CO₃, 40 µL 30% sucrose, and 5% SDS and were then vortexed at 3,000 rpm for 30 min at room temperature to extract total proteins. Insoluble proteins were removed by centrifugation at 12,000 g for 10 min at 4°C. The concentration of total membrane proteins in the supernatant was measured with a CB-X protein assay kit (G-Biosciences, USA). Proteins were separated by SDS-PAGE (4–20% precast polyacrylamide gel, Bio-Rad, USA) and transferred to nitrocellulose membranes. Primary antibodies of the D1 protein of PSII, Rieske iron-sulfur protein (RISP) of cytochrome b₆f complex, PsaA protein of PSI, and PsbO protein of the oxygen-evolving complex (OEC) were obtained from Agrisera (Sweden). Antigen-antibody complexes were visualized using an enhanced chemiluminescence substrate detection kit (Thermo Fisher Scientific, USA). Intensities of visualized protein bands were measured by using the program ImageJ (http://imagej.nih.gov/ij/).