Precast 4 20 sds page gels

Samples were loaded into precast 4–20%SDS-PAGE gels (BioRad) and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline, pH 7.4 containing 0.05% Tween 20 (PBST), at room temperature for 1 h, and were incubated with primary anti-barley oxalate oxidase 1 antibody (Origene, AP21356AF-N, rabbit polyclonal, 1:200 dilution) at 4°C overnight. Membranes were washed with PBST and a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP; Cell Signaling Technology, #7074, 1:1000 dilution) was incubated at room temperature for 1 h, and then washed with PBST. HRP signal was activated using SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and Images were taken using an ImageQuant LAS 4000 imager (GE Healthcare). The antibodies were diluted to their appropriate ratio according to manual instructions. Experiments were performed in triplicate and a representative blot was shown. Original uncropped and unadjusted blot and gel images are shown in S1 File.

After the separation of proteins on precast 4–20% SDS-PAGE gels (Bio-Rad), they were transferred onto methanol-activated polyvinylidene difluoride (PVDF) membranes. Nonspecific binding was blocked with 5% milk in phosphate-buffered saline and 0.1% Tween 20 (PBST) for 1.5 h at room temperature. The PVDF membranes were then incubated with primary antibodies (1:1000 dilution; catalog no. A8592, Sigma-Aldrich) against the FLAG tag for 1.5 h at room temperature and washed three times for 15 min with PBST. Then PVDF membranes were developed using the Western horseradish peroxidase (HRP) substrate for enhanced chemiluminescent detection (catalog no. 32132; Thermo Fisher Scientific). As reported before^{47 (link)}, the relative intensity of each immunoreactive band was estimated with ImageJ, where a linear response was confirmed over the range used.