COP1 | E3 ubiquitin-protein ligase COP1

Total protein extracted freshly from 15 seedlings (7-day-old grown under short day condition with 8h light/16h dark) with 2x Laemmli sample buffer (Biorad, 120 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 200 Mm dithiothreitol), and then denatured at 95°C for 10 min. Proteins were separated on pre-cast SDS-polyacylamide gels with a 7.5% acrylamide and blotted 7 min to nitrocellulose membranes (Biorad Trans-Blot Turbo RTA Nitrocellulose Transfer Kit), using semi-dry transfer. Blot was blocked with EveryBlot Blocking Buffer (Biorad) for 10 min at RT with agitation. Bolt was incubated in the anti-COP1 antibody at a dilution of 1:1000 for 4 °C/ON with agitation. The primary antibody solution was decanted, and the membrane was first washed briefly once (5 s) and then for 15 min, followed by 3 additional washings of 5 min in 1x TBS-T (without blocking agent) with agitation. Blot was incubated in Agrisera matching secondary antibody (Goat anti-Rabbit IgG, HRP conjugated, AS09 602) diluted to 1:25000 for 1h at RT with agitation). The blot was washed as above and developed for 2~3 min with chemiluminescent detection (AgriseraSuperBright, AS16 ECL-S-10), according to the manufacture’s instructions. The Exposure time was 20 seconds.​