Protein size markers

The phage particles were purified using CsCl and concentrated. The suspension was mixed with protein sample buffer, boiled for 10 min, and then separated on a 12% SDS-PAGE gel. Protein size markers were purchased from Bio-Rad, and the protein bands were visualized using InstantBlue (Expedeon Protein Solutions, Ltd., Cambridge, UK). For better visualization of low-copy structural proteins, gels were stained using the silver staining method^{59 (link)}. The identity of the virion proteins was elucidated using LC/MS/MS (Energenesis Biomedical Co., Ltd., Taiwan).

A previous report outlined the process of protein extraction from rice powder and the method of moderate denaturation to achieve higher resolution SDS-PAGE results (Takahashi et al. 2019 (link)). SDS-PAGE was performed using 15% polyacrylamide gels (ATTO, Tokyo, Japan) with protein-size markers (Bio-Rad, Hercules, MA, USA). After separation, the proteins were stained with 0.1% Coomassie Brilliant Blue (CBB) R-250 (Nacalai tesque, Kyoto, Japan). The gel was destained for at least 3 h before being scanned directly. Quantity One software ver. 4.6.9. (Bio-Rad) was used to determine the intensity of TP intensity in a lane, glutelin acidic subunit, glutelin basic subunit, and prolamin. The ratios of glutelin (G) / TP, prolamin (P) / TP, and other proteins / TP were calculated as follows:

SDS–PAGE was performed using 15% polyacrylamide gels (ATTO) with protein size markers (Bio-Rad, Hercules, MA, USA). After separation, the proteins were stained with 0.1% Coomassie brilliant blue (CBB) R-250. The gel was destained for at least 3 h before being directly scanned using an EPSON GT 900 scanner (Nagano, Japan).
For immunoblot analysis, proteins were transferred from gels to 0.22-μm pore PVDF membranes (Whatman) at 12 V, 60 mA, for 30 min. The membrane was soaked in blocking buffer (Nacalai tesque, Inc., Kyoto, Japan). After incubation with target primary antibodies (1/1000–1/8000 dilution), the membrane was washed three times with PBS-containing Tween 20. The membrane was incubated with secondary antibodies conjugated with HRP before being washed a further three times with PBS-containing Tween 20. Proteins were detected by chemical luminescence (ECL prime, GE Healthcare Japan, Tokyo, Japan) using either an LAS 1000 (GE Healthcare) or ChemiDoc™ MP imaging system (Bio-Rad). QuantityOne software ver. 4.6.9. (Bio-Rad) was used to determine the intensity of detected protein bands.