Ara h1, clone 17
AS16 3977 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arachis hypogaea
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Thirty (30) µg of total protein extracted freshly from defatted lightly roasted peanut flour with borate buffered saline (BBS) solution (100 mM H3BO4, 25 mM Na2B4O7, 75 mM NaCl, and pH 8.6) for 1 hr with constant stirring at 4°C. Samples were denatured with NuPAGE™ LDS sample buffer containing 50 mM DTT at a 1:4 (v/v) ratio and incubation at 70°C for 5 min. Samples were separated on Novex™ 10-20% Tricine Protein Gels and blotted 7 minutes to nitrocellulose using iBlot dry transfer system. The blot was blocked with 5% milk in phosphate buffered saline (pH 7.4) with 0.1% tween-20 (PBST) for 1h/RT with agitation. The blot was incubated in the primary antibody at a dilution of 1: 1,000 for 1h/RT with agitation in PBS-T with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed 3 times with 10mL for 5 min in PBST at RT with agitation. The blot was incubated with Donkey anti-Rabbit IRDye 680RD secondary antibody (LI-COR, Nebraska, US) diluted to 1:10,000 in PBST for 1h/RT with agitation. The blot was washed as above, scanned on a LI-COR Odyssey, and images were collected with the 680nM channel.