AS09 566 | Clonality: Polyclonal | Host: Hen | Reactivity: Arachis hypogaea (peanut)
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Arachis hypogaea protein extract
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 for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1,000 for 1h/RT with agitation in TBS-T with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed 3 times for 5 min in TBS-T at RT with agitation. The blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS10 1489) diluted to 1:25,000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 5 min with AgriseraECLBright. Images of the blots were collected using a CCD imager and Quantity One software (Bio-Rad). Exposure time was 20 seconds.
Peanut (Arachis hypogaea) belongs to the legume family. Dietary substances from peanuts can be a cause of allergi reaction in estimated 0.4-0.6% of population.
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