Protein deglycosylation.

Protein deglycosylation were analyzed using a Protein Deglycosylation Mix II set (NEW ENGLAND Biolabs, #P6044) following the manufacture’s protocol. In brief, under native conditions, the 100 μg of glycoproteins (BSA or RBD-Ferritin) were dissolved into 40 μl H₂O then add 5 μl 10×Deglycosylation Mix Buffer 1. Next, add 5 μl Protein Deglycosylation Mix II, mix gently and incubate reaction at 25°C RT for 30 min. After that, transfer reaction to 37°C, incubate for 16 h. While under denaturing condition, 100 μg of glycoproteins (BSA or RBD-Ferritin) were dissolved into 40 μl H₂O then add 5 μl of Deglycosylation Mix Buffer 2, incubate at 75°C for 10 min, cool down. Next, add 5 μl Protein Deglycosylation Mix II, mix gently and incubate reaction at 25°C RT for 30 min. After that, transfer reaction to 37°C, incubate for 1 h. Finally, assess the extent of deglycosylation by mobility shifts on SDS-PAGE gels. The nondenatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.