Pngase f

The FF-purified IgG and total proteomes were deglycosylated as described previously with minor modifications [36] . Briefly, prior to enzymatic release of N-linked glycans, equal amounts of purified FF IgG corresponding to 3 µg of isolated proteins were dried in vacuum by a vacuum concentrator (ThermoFisher, USA). Dried purified FF IgG samples were then re-suspended in 10 µl of 5x PBS (Sigma Aldrich, USA) which was followed by addition of 20 µl of 2 % SDS (BioRad, USA) to each sample. The samples were then incubated on 60°C for 30 min. After incubation, 12 µl of PNGaseF release mixture (1:1 4% NP-40: 5x PBS containing 0.75 units of PNGase F, Promega USA) was added to each sample which was followed by an 18 h incubation at 37°C. Similarly, the same amount of remaining FF samples corresponding to 70 µg FF total proteomes were precipitated overnight in 4 volumes of ice-cold acetone (at -20°C; Sigma Aldrich). After precipitation of proteins, the samples were resuspended in 12 µl with 5x PBS followed by addition of 12 µl of 2% SDS to each diluted sample. The samples were then incubated for 30 min at 60°C. Afterwards, 12 µl of PNGaseF release mixture was added to each sample which was followed by incubation of samples for 18h at 37°C. The released FF purified IgG and FF total proteomes N-glycans were stored at -80°C prior to ethyl-esterification procedure.