BZR1 | Brassinazole resistant 1 (Oryza sativa)
AS16 3219 | Clonality: Polyclonal | Host: Rabbit | Reactivity:
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store at 4°C; make aliquots to avoid working with a stock. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from liquid material adhering to the cap or sides of the tubes.
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Total protein from Oryza sativa rice (CV. 9311) flag leaf at the tillering stage was ground into a fine powder in liquid nitrogen. An 800 ul aliquot of extraction buffer [62.5 mM TRIS-HCl (pH 7.4), 10% glycerol, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5% (v/v) b-mercaptoethanol] was added to each 300 mg powder sample. The mixture was vortexed and then chilled on ice for 10 min. Samples were centrifuged at 12 000 rpm for 10 min at 4℃, and the supernatant was collected and stored at –70 ℃. The protein concentrations of the rice samples were determined using the Bradford method (Bradford, 1976). 20 µg of protein was separated on 12 % SDS-PAGE and blotted 1h to PVDF.
Blots were blocked with for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer’s instructions. Exposure time was 2 min