Simultaneous immunodetection of ATPB (AS05 085), LHCSR3 (AS14 2766) and COXIIB (AS06 151) in Chlamydomonas reinhardtii whole cell extracts. Whole cell extracts (30 µg protein) were loaded on Biorad 4-20% Criterion TGX Precast Mini protein gels. Gels were run at 85 V. For electro-blotting, wet transfer onto nitrocellulose membrane was performed (105 V for 1 h). Overnight blocking with 5% milk in TBST in the cold room. Sequential 2 h-long incubation with mixed primary antibodies each at a dilution of 1:20 000 in TBS-t, followed by incubation with anti-rabbit antibody and development with ECL. Image was taken using Chemidoc MP Imaging system.
Lane from the left: samples from dim light; Lane to the right: samples after 4h at 250 µmol photons m-2 s-1.
Courtesy of Msc. Angeliki Tsichla, Petroutsos and Finazzi lab, CEA Grenoble, France
Primary antibodies of similar titer, made in the same host, can be used in simultaneous western blot.