Anti-Thioredoxin-like protein HCF164, chloroplastic

Arabidopsis thaliana Col0 thylakoids were extracted in isolation buffer (330 mm Sucrose, 25 mm HEPES-KOH, pH 7.4, 10 mm MgCl₂, and 10 mm NaF) and detarud with Laemmli buffer at 65C for 10 min. Proteins were separed on 12 % SDS-PAGE and blotted  1 h on a PVDF membrane using semi-dry transfer. Blot was blocked with 8 % milk for 1 h at RT. Blot was incubated in the primary antibody at a dilution of 1: 3 000 overnight at 4°C with agitation in TBS-T with 1% milk. The antibody solution was decanted and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 from Agrisera) diluted to 1:25 000 in  for 2h at RT with agitation. The blot was washed as above, incubated in chemiluminescent detection reagent for 5 min and developed on film with 1 min exposure.