Quantifying Rice Leaf Proteins

Rice total protein was extracted (Chye et al., 1999 (link); Liao et al., 2014b (link)) from 1-week-old fresh rice leaves and protein concentration was measured by the Bradford assay (Bradford, 1976 (link)). Proteins (20 μg) were resolved on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pall). Western blot analysis was performed as described previously (Lung et al., 2017 (link); Liao et al., 2018 (link)). The blots were cross-reacted with rabbit polyclonal anti-GFP (1: 5000, A6455; Invitrogen) for OsACBP4promoter:OsACBP4:GFP transgenic lines or rabbit polyclonal anti-RFP (1:7000, R10367; Invitrogen) for OsACBP5promoter:OsACBP5:DsRED transgenic lines at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:50,000; Sigma-Aldrich) at room temperature for 1 h. Cross-reacting bands were detected by the Amersham ECL Prime Detection Reagent (GE Healthcare).