Evaluation location: Illinois, United States
Accessions were screened to identify accessions with low or no P34 expression. Seeds were weighed to 0.5 g for the assay for each accession was used as starting material to extract total protein as the allergen of interest is expressed highly in seeds. because of their smaller size.Pulverized seed from each accession was added to 1mL TBS-T buffer [0.05MTris, 0.5MNaCl,pH7.4 with 0.05%(v/v)Tween 20 (polyoxyethylene sorbitan monolaurate)] and incubated for 30 min on a shaker (200 rpm) at room temperature. Immuno-blot assay of the seed extracts was done by a rapid and large scale method as follows: A 10-mL aliquot of each sample extract was dot-blotted on a nitrocellulose membrane (0.45 mm; Fisher Scientific) with a P34 positive control sample (cv. Kunitz). To check for equal loading of the samples on the blots, membranes were stained with Ponceau S solution (Sigma, St. Louis, MO) for 1 min and rinsed in double distilled water. The membrane was incubated in TBS buffer (0.05 M Tris and 0.5 M NaCl, pH 7.4) for 5 min and blocked (10% nonfat dry milk powder in TBS buffer) for 1 h followed by rinsing twice in TBS buffer for 5 min each. The membranes were incubated in P34 protein specific monoclonal antibody P4B5 (diluted 1: 5000 in TBS-T buffer with 0.1% milk powder) for 1 h. The membranes were washed extensively in TBS-T buffer and incubated in the secondary antibody [antimouse IgG (Fab specific) alkaline phosphatase conjugate (Sigma)] diluted 1: 10 000 in TBS-T buffer with 0.1% milk powder for 1 h and rinsed in TBS-T buffer and TBS buffer once each. The P34 protein was detected by a color substrate system [BCIP/NBT: final concentrations 0.02% (w/v) 5-bromo-4-chloro-3-indolyl phosphate and 0.03% (w/v) nitro blue tetrazolium in 70% (v/v) dimethylformamide] (Sigma).