Albumin from chicken egg white

32 female Balb/c mice (Core Unit for Biomedical Research, Division for Laboratory Animal Science and Genetics, Himberg, Austria) were randomly divided in 4 groups of 8 and were kept under conventional condition (12 h light/dark cycle at 22 °C). 2 out of the 4 groups (a and n) were fed with LasQCdiet^®Rod16-A (Soest, Germany), composed of a higher amount of long-chain polyunsaturated FAs due to the added linseed oil and lack of soybean product (soy-free feed; Table 1). The other 2 groups (A and N) were fed with ssniff fortified V1534-300 (Soest, Germany) consisting of a lower percentage of long-chain polyunsaturated FAs and soybean products (soy-containing feed; Table 1). All groups had unlimited access to water and food.
Animals were treated according to European Union guidelines of animal care and the protocol has been approved by the local Ethics committee of the Medical University of Vienna and by the Austrian Federal Ministry of Science and Research with the approval number BMWFW-66.009/0182-WF/V/3b/2017.
Our model allergen of interest was Ovalbumin (OVA; Albumin from chicken egg white, Sigma Aldrich, Vienna, Austria, #A5503). As a control the mice were kept naive. For all treatments OVA was freshly dissolved in water to gain requested concentrations.

Platelets were diluted to a final concentration of 2 × 10⁴ cells/mL in cell culture medium (DMEM, Sigma-Aldrich, Vienna, Austria), pipetted onto a glass slide and incubated for 15 min. Non-adhered cells were washed away with PBS (phosphate-buffered saline). For DIC microscopy, cells were fixed using 4% paraformaldehyde in distilled water and imaged. The actin cytoskeleton was visualized using Alexa Fluor 647 phalloidin and Alexa Fluor 488 phalloidin (Cell Signaling Technology, Leiden, The Netherlands), respectively. Platelet actin staining was performed in Cytoskeleton buffer with sucrose (CBS) containing 10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl₂, 2 mM EGTA, and 0.32 M sucrose according to a protocol of Louise Cramer (MRC Laboratory for Molecular Cell Biology, UCL, London, UK) [24 (link)]. Briefly, platelets were fixed using 4% paraformaldehyde in CBS for 20 min at room temperature, permeabilized in 0.5% Triton X-100 with CBS, blocked in 10% albumin from chicken egg white (Sigma-Aldrich, Vienna, Austria) and stained for 20 min with 66 nM fluorophore conjugated to phalloidin. For CD62p labeling, fixed cells were stained with 1 µg/mL anti-CD62p antibody marked with Alexa 647 (BioLegend, San Diego, CA, USA) for 10 min.

Maize seed proteins were sequentially extracted with water (albumins), 5% (w/v) NaCl (globulins), 75% (v/v) ethanol (Osborne prolamins) and 0.25% (w/v) NaOH (Osborne glutelins), as previously described [42 (link)].
The supernatants were dialyzed overnight (except ethanol fraction) using SnakeSkin pleated dialysis tubes with a cutoff of 3.5 kDa (Thermo Scientific). Ethanol fraction and all the other dialyzed protein fractions were subsequently lyophilized.
For gel electrophoresis the obtained pellets were dissolved in solubilisation buffer (2 M thiourea, 0.4% (v/v) triton X-100, 7 M urea, 4% (w/v) CHAPS, 1% IPG buffer 3–11, 60 mM DTT) and protein concentration measured according to Ramagli [43 (link)], with albumin from chicken egg white (Sigma) as standard.
At the end of this step we had 36 protein pellets (4 protein fractions × 3 ears/ plants × 3 maize lines) (Additional file 2: Figure S1).

Albumin from chicken egg white, α-lactalbumin from bovine milk and κ-casein from bovine milk were obtained from Sigma (Zwijndrecht, the Netherlands). Papain enzyme from Carica papaya (10 mg/mL) and TFA was purchased from Merck (Hohenbrunn, Germany). The protein standard solutions were prepared by dissolving 1 mg of the respective protein in 1 mL demineralized water (Millipore, Amsterdam, the Netherlands) in individual Eppendorf tubes. Standard solutions were pre-incubated with 30 µL of papain solution (1 mg/mL) at 65 °C for 5 min. The enzyme-to-protein ratio was 1:33. The sample was incubated overnight (16 h) at 37 °C. Enzyme deactivation was then performed by heating the solutions to 95 °C for 5 min.

Low-molecular-weight chitosan (50–190 Da), sodium tripolyphosphate, polycaprolactone ~ 14,000 Da (PCL), isopropyl β-D-1-thiogalactopyranoside (IPTG), LB medium, albumin from chicken egg white (Ova), urea, Tris buffer and Tween 80 were obtained from Sigma-Aldrich (USA). Chitosan (> 95% deacetylate, high quality ChitoClear) was obtained from Primex (Iceland). Fluorescein isothiocyanate (FITC) MW 389,382 g/mol was obtained from Thermo Fisher (Czech Republic). Hyaluronic Acid HYSILK (Mw ~ 150–350 kDa) was purchased from Contipro (Dolni Dobrouc, Czech Republic). CpG ODN and Poly (I:C) were from InvivoGen (France).

Albumin from chicken egg white (OVA), fetal bovine serum (FBS), penicillin, Roswell Park Memorial Institute 1640 (RPMI 1640) medium, and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Purified rat anti-mouse IgE, biotin rat anti-mouse IgE, goat affinity purified antibody to mouse IgG, and biotin-XX goat anti-mouse IgG₁ were purchased from BD Biosciences (Franklin Lakes, NJ, USA), MP Biomedicals (Santa Ana, CA, USA), and Life Technologies (Carlsbad, CA, USA), respectively.