Deglycosylation of Influenza HA Protein

Deglycosylation was performed using PNGase F or Endo H enzyme (New England BioLabs, MA) as previously described (57 (link)). In brief, virus samples were predenatured and then treated with or without the PNGase F for 15 min at 50°C or Endo H enzyme for 1 h at 37°C according to the manufacturer's instructions. Then samples were heated at 100°C for 10 min and then separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), and subsequently incubated with a primary antibody specific for H5N1 influenza HA (1:2,000, 11689-T54, Sino Biological). The secondary antibody used was horseradish peroxidase (HRP)-conjugated antirabbit IgG (1:10,000, A0208, Beyotime). HRP was detected using a Western Lightning chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany) according to the manufacturer's protocols.

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Sun H., Deng G., Sun H., Song J., Zhang W., Li H., Wei X., Li F., Zhang X., Liu J., Pu J., Sun Y., Tong Q., Bi Y., Xie Y., Qi J., Chang K.C., Gao G.F, & Liu J. (2022). N-linked glycosylation enhances hemagglutinin stability in avian H5N6 influenza virus to promote adaptation in mammals. PNAS Nexus, 1(3), pgac085.

Publication 2022

PNGase F Endo H enzyme PVDF) membrane horseradish peroxidase (HRP)-conjugated antirabbit IgG Western Lightning chemiluminescence kit

Antibody Biological Chemiluminescence Endo Endo 1 Enzyme H5n1 Horseradish peroxidase Influenza Membrane Pngase f Polyvinylidene difluoride Sds page Sino Virus

Corresponding Organization : China Agricultural University

Other organizations : Chinese Academy of Sciences, Tsinghua University, University of Nottingham, National Institute for Viral Disease Control and Prevention, Chinese Center For Disease Control and Prevention

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Variable analysis

independent variables

Enzyme used for deglycosylation (PNGase F or Endo H)

dependent variables

Molecular weight of influenza HA protein after deglycosylation, as observed on SDS-PAGE gel

control variables

Virus samples
Predenaturation of virus samples
Incubation time and temperature for deglycosylation (15 min at 50°C for PNGase F, 1 h at 37°C for Endo H)
Heating of samples at 100°C for 10 min
SDS-PAGE gel electrophoresis
Transfer to PVDF membrane
Primary antibody (1:2,000 dilution, anti-H5N1 influenza HA)
Secondary antibody (1:10,000 dilution, HRP-conjugated anti-rabbit IgG)
Western Lightning chemiluminescence detection

controls

Positive control: Virus samples treated with PNGase F or Endo H enzyme
Negative control: Virus samples without treatment with PNGase F or Endo H enzyme

Annotations

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