Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

To determine purity of both yolk- and serum-derived IgY, SDS-PAGE was conducted under reducing conditions using 12% polyacrylamide gel (NuSep Inc., Germantown, MD, USA) with a Novex Mini-Cell (Invitrogen, Carlsbad, CA, USA). Briefly, the sample was mixed with sample buffer and incubated for 5 min at 100°C. A total of 10 μl of sample was loaded into each well. Precision Plus Dual Color Pre-stained protein standard (10-250kDa) (Bio-Rad, Hercules, CA, USA) was used as a molecular weight marker. The protein bands were visualized with Protein Fixative (Ward’s Science, Rochester, NY, USA). The gel was imaged using a standard camera; the original gel image can be found in the S1 Raw image. Specificity of IgY binding to purified S1 glycoprotein was demonstrated by dot-blot (S2 Fig).