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Protein Deglycosylation

LW Lyn M. Wise
GS Gabriella S. Stuart
KS Kevalee Sriutaisuk
BA Brooke R. Adams
CR Christopher B. Riley
CT Christine L. Theoret
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To compare the glycosylation states of eIL-10 and hIL-10, purified protein (500 ng) was diluted in 0.05 M sodium phosphate (pH7) containing 0.1% sodium dodecyl sulfate and 20 mM 2-mercaptoethanol and boiled for 5 min. The mixture was cooled on ice and Tween 20 was added to 0.75%. Either five units of N-glycosidase F (Roche, Mannheim, Germany) or no enzyme was added, then the mixtures incubated at 37°C for 3 h. The deglycosylated proteins were then resolved by SDS-PAGE and visualized by western blotting using an anti-FLAG Peroxidase antibody (M2, mouse monoclonal, Sigma-Aldrich).

Copyright and License information:  ©2020 Wise, Stuart, Sriutaisuk, Adams, Riley and Theoret.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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