Western blot incubation of few primary antibodies at the same time

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 85V. For electro-blotting wet transfer onto nitrocellulose membrane was performed (105 V for 1h). Overnight blocking with 5% milk in TBST in the cold room. Sequential 2h-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 and made in the same host can be used in simultaneous western blot.
2017-04-24 @ 14:33:17