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. 2021 Aug 12;13(8):1595. doi: 10.3390/v13081595

Figure 3.

Figure 3

Gel filtration chromatography and glycan detection of plant-produced gRBD or dRBD proteins. Profiles of plant-produced gRBD (A) and dRBD (B) proteins, eluted from Sephacryl® S-200 HR column. The column was equilibrated with 50 mM phosphate buffer (with 150 mM NaCl, pH 7.4). The plant-produced dRBD and gRBD proteins (0.25 mg), purified using FLAG affinity chromatography, were loaded onto column. Gel filtration was performed with ÄKTA start using 60 cm × 16 mm column (cat. no. 19-5003-01, GEHealthcare, Chicago, IL, USA), packed with Sephacryl® S-200 HR (cat. no. 17-0584-10, GE Healthcare). (C) SDS-PAGE analysis of plant-produced gRBD and dRBD proteins eluted from Sephacryl® S-200 HR column. (D) Glycan detection in gRBD and dRBD proteins. Protein (250 ng) from each sample (eluted from Sephacryl® S-200 HR column) was separated on a 10% SDS-PAGE gel followed by in-gel glycan detection using the Pro-Q Emerald 300 glycoprotein staining kit. Stained proteins were visualized by UV illumination. M—CandyCane glycoprotein molecular weight standards (Molecular Probes), 250 ng of each protein per lane; gRBD—glycosylated RBD; dRBD—deglycosylated RBD; gPA83—glycosylated PA83; dPA83—deglycosylated PA83. (E) Western blot analysis of the same sample using the anti-FLAG antibody. Purified Endo H protein (25, 50, and 100 ng) and gRBD or dRBD proteins (50 ng) were loaded into wells.