Mini trans blot cell wet blotting system

Proteins separation of PSII-enriched membranes isolated from WT and cia3 was carried out by electrophoresis under denaturing conditions in a 16% polyacrylamide gel [82 (link)] in Mini-PROTEAN 3 Cell (BioRad). The samples were loaded on a gel at an equal amount of Chl content equal to 1.5 μg per track unless otherwise indicated. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane (Amersham, Protran, 0.45 μm NC) using the Mini Trans-Blot Cell wet blotting system (BioRad). The membrane was incubated overnight at 4 °C with anti-rabbit primary antibodies against D1, PsbO, Lhcb1, Lhcb2, and CAH3 proteins produced by Agrisera (Sweden) (AS11 1786, AS06 142-33, AS01 004, AS01 003 and AS05 073, respectively). Donkey anti-rabbit antibodies labeled with horseradish peroxidase (GE Healthcare) were used as secondary antibodies in a dilution of 1:5000. The antibody-antigen conjugates were detected by a Pierce ECL Plus Western Blotting kit (Thermo scientific) and the gel documentation system ChemiDoc (BioRad). Quantification of bands on the blots was performed by ImageJ software (version 1.53a, National Institutes of Health, Bethesda, MD, USA).

The total protein was isolated using RIPA cell lysis buffer supplemented with protease inhibitors (Complete Mini, Roche, Basel, Switzerland). SDS-PAGE was performed using the Mini-PROTEAN^® Tetra Cell electrophoresis chamber (BioRad, Hercules, CA, USA). The PageRuler Prestained NIR Protein Ladder (Thermo Fisher Scientific) was used. Separated proteins were blotted onto a nitrocellulose membrane (Amersham Protran 0.2 NC, Sigma-Aldrich) using the Mini Trans-Blot^® Cell wet blotting system (BioRad). Membranes were incubated with primary antibodies (rabbit anti-rat p75 NGF receptor antibody; mouse anti-rat beta-actin antibody (both Abcam, Cambridge, UK)) overnight at 4 °C, and infrared-labeled secondary antibodies (IRDye 800CW donkey anti-goat, Li-Cor; IRDye 680CW goat anti-mouse (Li-Cor Biosciences, Lincoln, NE, USA)) for one hour at room temperature. Visualization was performed using the Odyssey^® Infrared Imaging System.