Anti-BZR1 | Brassinazole resistant 1 (Oryza sativa)

The total protein content of 7-day-old rice seedlings was extracted using an extraction buffer containing 50 mM Tris pH 7.5, 150 Mm NaCl, 0.5% Triton X-100, and a complete protease inhibitor (Sigma) (0.2 g of sample plus 100 ml of extraction solution). Cellular debris was removed by centrifugation at 4℃, 12,000rpm for 5 minutes, and the supernatant was mixed with the 4×Laemmli buffer (250 mM Tris pH 6.8, 8% SDS, 40% glycerol, 4% β-mercaptoethanol, 0.01% bromophenol blue) in a ratio of 3:1. After mixing, it is denatured at 95℃ for 5 minutes, followed by centrifugation at 12,000 rpm for 5 minutes. The supernatant is then for Western blotting. Proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes, and then, the PVDF membrane was blocked with 5% milk in TBST (0.8% NaCl, 0.02% KCl, 0.3% Tris, pH 7.4, and 0.05% Tween 20) for an hour at room temperature. Excess milk was washed off with TBST. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 3 h/RT with agitation in TBST. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 15 min in TBST at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG) diluted to 1:5 000 for 2 h/RT with agitation. The blot was washed three times for 15 min in TBST. Exposure time was 120 seconds at full resolution (Bio-Rad, ChemiDoc system).