20 or 40 µg of total protein from Arabidopsis thaliana (WT) leafs or ferredoxin mutant fd2 were separated on 15 %  acrylamide gel with 6 M urea. Filters were blotted on PVDF, blocked (1 h) with 5 % milk, incubated with 1: 1000 anti-ferredoxin (over night in 4ºC) in 1 % milk/TBS-T) followed by incubation with 1: 10000 secondary antibody (2 h) coupled to HRP and visualization with standard ECL. 

20 or 40 µg of total protein from Arabidopsis thaliana (WT) leafs or ferredoxin mutant fd2 were separated on 15 %  acrylamide gel with 6 M urea. Filters were blotted on PVDF,5 µg of soluble protein extracts from Chlamydomonas reinhardtii strain CC-4532 were separated on 15 % SDS-PAGE and blotted at 4 W for 30 min to nitrocellulose membrane (0.1 µm pore size) using semi-dry transfer. The blot was blocked with 1 % non-fat dry milk overnight at room temperature (RT) with agitation. The blot was incubated in the primary antibody solution at a dilution of 1: 10 000 in PBS-T+1% milk for 2h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. The blot was incubated in secondary antibody solution (anti-rabbit IgG-AP conjugated) diluted to 1: 300 in PBS-T+1% milk for 1h at RT with agitation. The blot was washed as above and developed with exposure time of 2.5 minutes.

Courtesy of Dr. Marcus Miethke, Department of Chemistry & Biochemistry, UCLA