PhyB | Phytochrome B (dicots)

30 μg/well of total protein extracted from 4-days-old Arabidopsis thaliana seedlings with extraction buffer (65 mM Tris/HCl pH 6,8; 4 M Urea; 10% glycerol; 3% SDS; 0.05% bromphenol blue; 20 mM DTT). Protein extracts were stored overnight at -20°C. Prior to separation on SDS PAGE, extracts were thawed and incubated for 5 min at 65°C followed by centrifugation and were separated on 9% SDS-PAGE and blotted 2 h to PVDF membrane (Immobilon-P pore size 0.45 μm) using wet transfer. Blot was blocked with 5% milk powder in TBS-T for 1 h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 in TBS-T ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed with TBS-T briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602) diluted to 1:25 000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 5 min with AgriseraECLSuperBright with Fusion SL (Peqlab) luminescence camera system. Exposure time was 8 minutes at full resolution.

Courtesy of Dr. Andreas Hiltbrunner, Freiburg University, Germany