Rapid peptide n glycosidase pngase f

Chemical reagents and solvents were from Sigma-Aldrich (St.Louis, MO). Oligosaccharide standards (A1F, NA2, NA2F, M3N2, M3N2F, N2F) were purchased from ProZyme (Hayward, CA). Rapid Peptide-N-Glycosidase (PNGase F), Remove-iT^® Endo S, α1-2,4,6 Fucosidase, and Proteinase K were from New England Biolabs (Ipswich, MA). Rabbit and human serum IgG antibodies were obtained from Sigma-Aldrich.

SDS-PAGE was performed to characterize live, inactivated, and inactivated split virus samples. Before running the SDS-PAGE, samples were deglycosylated with rapid peptide:N-glycosidase (PNGase) F (New England Biolabs) according to the manufacturer’s instructions for a better resolution of the bands. After deglycosylation, 20 μL of sample was mixed with 2X Laemmli buffer (BioRad) containing 50 mM NuPAGE sample reducing agent (dithiothreitol, Thermo Fisher Scientific). Samples were then incubated for 10–15 min at 90°C–95°C and run on a 4%–20% precast polyacrylamide Mini-PROTEAN TGX gel (BioRad) for 1 h at 120 V (30 μL/well). The gel was stained with SimplyBlue SafeStain (Novex) O/N, and destained in distilled water. Images were taken in a Chemidoc MP Imaging System using the Image Lab software (BioRad).