Purified starch granules were resuspended at a concentration of 33 mg/ml in SDS–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer [50 mM tris-HCl (pH 6.8), 2% (w/v) SDS, 100 mM dithiothreitol, 3% (v/v) glycerol, and 0.005% (w/v) bromophenol blue] and heated at 95°C for 10 min. Following a 60-min centrifugation, samples were loaded onto a 10% SDS-PAGE gel (Criterion TGX Precast Gel, Bio-Rad) and electrophoresed in tris-glycine-SDS running buffer. Proteins were transferred to low-fluorescence polyvinylidene difluoride membranes using the Trans-Blot Turbo Transfer System (Bio-Rad) and then incubated for 1 hour in blocking solution [5% (w/v) milk powder in 1× tris-buffered saline (TBS)]. The membrane was then incubated for 18 hours at 4°C in primary antibody diluted in blocking solution with 0.1% (v/v) Tween 20. The GBSS antibody was raised in rabbit (35) and used at a 1:1000 dilution. The membrane was washed four times for 5 min, each in 1× TBS and 0.1% (v/v) Tween 20 (TBST) at 20°C, before incubation in secondary antibody [donkey anti-rabbit conjugated to IRDye 800CW (LI-COR) used at 1:10,000 dilution] for 1 hour at 20°C. The membrane was washed four times for 5 min each in 1× TBST and then twice for 5 min in 1× TBS. The protein was visualized by infrared fluorescence using an Odyssey CLx Imaging System (LI-COR).

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