UVR8 | Ultraviolet-B receptor UVR8

20 µg/well of total protein extracted from liquid N2-frozen leaves of adult plants using hot extraction buffer containing 65 mM Tris-HCl, pH 7.8, 4 M urea, 5% (w/v) SDS, 100 mM dithiothreitol, 15% (v/v) glycerol, and 0.05% (w/v) bromophenol blue and denatured at 95°C for 5 min. Samples were separated at RT on 10% SDS-PAGE and blotted for 2 h to PVDF (pore size of 45 µm), using wet transfer in the cold. Blot was blocked with 5% milk 1h/RT with agitation. The blot was incubated in the primary antibody at a dilution of 1:1000 in TBS-T during overnight (16 h) at 4 °C with agitation. The antibody solution was decanted, and the blot was rinsed briefly, then washed 5 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 Agrisera) diluted to 1:10 000 in TBS-T for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AS16 ECL-S-10 AgriseraSuperBright. Exposure time was 0.6 s (iBright, 3x3 binning).

Courtesy of Dr. András Viczián,Institute of Plant Biology. Biological Research Center Szeged Hungary