Chicken anti-Rabbit IgG (H&L), DyLightŪ 488 conjugated
AS10 831 | clonality: polyclonal | host: Chicken | reactivity: Rabbit IgG (H&L)
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1:20 - 1:2,000 for most applications
to be added when available
Samples of Arabidopsis thaliana (2), Olea europaea (3), Lilium Longiflorum (4), Lupinus luteus (5) were ground in liquid nitrogen to a very fine powder using a mortar and pestle and resuspended in 1.5 ml of extraction buffer (4% SDS, 2% 2-mercaptoethanol, 2 mM PMSF, 100 mM Tris-HCl pH 8.5). The samples were incubated for 3 min at 80°C. Protein suspensions were clarified by centrifugation at 13,500 g for 10 min at room temperature and the resulting supernatants were used. Total proteins (25 µg per sample) were separated by SDS-PAGE on CriterionTMTGXTM Precast Gel (Bio-Rad, USA) using CriterionTM Cell apparatus (Bio-Rad). Proteins were electroblotted onto a PVDF membrane using Trans-Blot® TurboTM Transfer Pack (Bio-Rad) in a Trans-Blot® TurboTM Transfer System (Bio-Rad). The membrane was blocked for 1 h in solution containing 1 % (w/v) non-fat dry milk in TRIS-buffered saline (TBS) buffer, pH 7.4. The membrane was incubated in the primary antibody at a dilution of 1: 1000 in TBS buffer containing 1 % (w/v) non-fat dry milk over night at 4°C with agitation. A DyLight 488 conjugated anti-rabbit IgG (AS10 831, Agrisera), diluted 1:2000 in TBS buffer for 2 h, served as the secondary antibody. The signal was detected in a Pharos FX molecular imager (Bio-Rad). Line 1 contains LOX protein from Sigma.
Courtesy of Dr. Agnieszka Zienkiewicz, CSIC, Spain
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