Abstract
This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is endogenously expressed in various cell types, including HCT116 cells, DT40 cells and mouse embryonic fibroblast cells. Partial deglycosylation by PNGase can be detected by faster migration of band in SDS-PAGE. You can compare tightness of the folding among wild-type and mutant proteins of interest. This method can be used with regular molecular and cell biology equipment, but applied only to glycoproteins.
Keywords: PNGase, Protein folding, Glycoproteins
Materials and Reagents
Equipment
Software
Procedure
Notes
Recipes
Acknowledgments
This protocol was adapted from and used in Ninagawa et al. (2015).
References
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