Zea mays Seed Protein Extraction and Western Blot

The protein was extracted from the developing Zea mays seeds according to the protocol described in [45 (link)]. SDS-PAGE was performed using a Vertical Electrophoresis System (Bio-Rad, Shanghai, China). Proteins were separated in SDS-PAGE on 10% acrylamide gels. Anti-rabbit antibodies (ZmPHO1) were generated according to the protocol described in [28 (link)]. After electrophoresis, the proteins in polyacrylamide gels were transferred to nitrocellulose membranes using an Electrophoretic Transfer Cell (Bio-Rad). The transfer buffer contained 10% running buffer, 20% methanol, and 70% water. After this membrane was incubated in blocking buffer (5% solution of skim milk in 1% TBST) for one hour on a rotator and then incubated overnight at 4 °C. A 15 µL quantity of ZmPHO1 antibody (affinity pure antibody) was added with (Anti-PHO1) 1:1000 dilution in blocking buffer, then incubated for 1 h on rotator at room temperature. The gel membrane was washed three times for 10 min each in 1% TBST. HPR secondary binding antibody Rabbit IgG was added with 1:2000 dilution in blocking buffer. The gel membrane was incubated for one hour. The gel membrane was washed three times for 10 min each in 1% TBST. A resolving solution was added according to the company or manufacturer’s instruction to photograph the bands’ results.