Comparative Photosynthetic Protein Analysis

WT and mutants were grown in TAP to early log phase under very low light (0~2 μmol photons m^-2 s^-1). Total protein was solubilized as previously described [29 (link)] and extracted using the methanol/chloroform method [55 (link)]. Each sample was quantified for protein concentration using BCA protein assay (Pierce) to load 5 μg protein in each lane of pre-cast SDS-PAGE Any kD Mini-PROTEAN gels (Bio-Rad), except for the dilutions of the WT samples. Proteins were transferred from the gel to a polyvinylidene difluoride membrane (Immobilon-FL 0.45 μm, Millipore) via wet-transfer for immunodetection. The primary polyclonal antibodies raised against AtpB (AS16 3976), D1 DE-loop (AS10 704), CP43 (AS11 1787), PsaA (AS06 172), and PsaD (AS09 461) were obtained from Agrisera. Donkey anti-rabbit IgG antibody with horseradish peroxidase (1:10,000; GE Healthcare) was used as a secondary antibody and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) chemiluminescent system on a ChemiDoc MP imaging system (Bio-Rad).