C3934

PPE (1 mg/mL), Ara h 1 (1 mg/mL), Ara h 2 (0.5 mg/mL), Ara h 3 (0.5 mg/mL) and Ara h 6 (0.5 mg/mL) in PBS were filtered through a 0.2 µm filter and individually loaded (50 µL) onto a Superdex 200 Increase 10/300 GL column (Cytiva, Uppsala, Sweden) connected to an ÄKTA pure 25 M system (Cytiva). Each protein sample was eluted at RT with PBS at an elution rate of 0.4 mL/min. The eluted proteins were detected by absorbance at 215 and 280 nm. The column was calibrated for molecular weight (MW) determination by applying a standard mixture consisting of 0.3 mg/mL ferritin (440 kDa; F4503, Sigma-Aldrich), 1 mg/mL conalbumin (79 kDa; C0880, Sigma-Aldrich), 1 mg/mL carbonic anhydrase (29 kDa; C3934, Sigma-Aldrich), 1 mg/mL cytochrome C (14 kDa; C2506, Sigma-Aldrich), 0.5 mg/mL vitamin B12 (1.3 kDa, V2876, Sigma-Aldrich). The presence and approximate quantification of Ara h 1, Ara h 2, Ara h 3 and Ara h 6 present in PPE was determined by overlapping peaks between PPE and the individual allergen followed by calculating area under the curve (AUC).