Anti-BAK1 | Brassinosteroid insensitive 1-associated receptor kinase 1 (monocots)

Samples:
WT - Oryza sativa wildtype 
bak1 - Oryza sativa BAK1 protein knockout mutant

Growth conditions and age of plant material were: The rice seeds were sterilized with 95% ethyl alcohol and washed five times with sterile water. The sterilized seeds were soaked in water for 2 days, and the uniformly germinated seeds were sown in bottomless 96-well PCR plates with appropriate spacing in the nutrient solution. They were then grown at 30℃ under white light at approximately 50 µmol m-2 s-1 for 14 h and then at 25℃ in a dark cycle for 10 h in a growth chamber for 10 days.

PM protein were performed according to previously described methods (Shen et al., 2017, Zhang et al., 2023). They were extracted by a Minute PM Protein Isolation Kit for Plants (Invent Biotechnologies). Samples were tested using antibodies against H+-ATPase (plasma membrane marker).The sample amount for each well is approximately 25ug.

Proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes for 90 min using wet transfer in the cold. and then, the PVDF membrane (pore size 0.2 µm) 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 1 hour at room temperature. Excess milk was washed off with TBST three times (5 min every time). Blot was incubated in the primary antibody at a dilution of 1: 1 000 overnight at 4℃ 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, AS09 602) diluted to 1:3 000 for 2 h/RT with agitation. The blot was washed three times for 15 min in TBST. Exposure time was 360 seconds at full resolution (Bio-Rad, ChemiDoc system).