D14 | Strigolactone esterase D14
AS16 3694 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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60 μg of soluble protein from seven-day-old Arabidopsis thaliana seedlings (grown under long day conditions on MS agar plates) extracted with PE buffer (50 mM TRIS pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Tween-20, 1 mM DTT, 1 mM PMSF, 1x Complete protease inhibitor (Roche)) and denatured with Laemlli buffer (including 125 mM DTT) at 95°C for 5 min. The samples were separated on 12% SDS-PAGE and blotted for 60 min to PVDF membrane using wet transfer. Blots were blocked with 2% BSA in TBST for 60 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 for 16 h in cold room with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 4 times for 5 min in TBST buffer at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:1000 in for 60 min at RT with agitation. The blot was washed as above and developed for 1 min with Clarity ECL substrate (Bio-Rad) using ImageQuant RT-ECL detection system (GE Healthcare). Exposure time was 1 min at medium resolution (1024*1024 pixels).
Dr. Mark Waters, The University of Western Australia
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