Verification of N-Linked Glycosylation of Proteins Isolated from Plant or Mammalian Cell Lines Using PNGase Enzyme.

N-glycosylation is a ubiquitous post-translational modification (PTM) that regulates protein folding, stability, and biological function. Accurate identification and validation of N-glycosylation are therefore critical for understanding how glycosylation modulates protein activity. Here, we present a robust workflow for analyzing protein N-glycosylation in both animal and plant systems using peptide-N ⁴-(N-acetyl-β-glucosaminyl) asparagine-amidase A and F (PNGase A and PNGase F). After enzymatic cleavage of the asparagine-linked N-glycans, samples are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB) to detect shifts in apparent molecular weight (MW) indicative of deglycosylation. Key steps include denaturing the protein to expose glycosylation sites, optimizing buffer conditions for PNGase F and A treatment, and comparing glycosylated vs. deglycosylated forms by electrophoretic mobility. A troubleshooting guide addresses common challenges, including incomplete deglycosylation and low transfer efficiency during WB, offering practical solutions to ensure reliable results. This protocol provides researchers with a standardized, cost-effective framework for investigating protein N-glycosylation in diverse systems, from cell lysates to purified proteins, in both animal and plant models. Key features • This method employs standard biochemical techniques, such as enzymatic digestion and SDS-PAGE, making it highly accessible and relatively quick to perform. • Successful deglycosylation results in a detectable downward shift in protein migration on an SDS-PAGE gel. This shift provides a visually confirmable indication of N-glycan removal. • Prior to engaging in mass spectrometry analyses, this approach serves as a rapid, cost-effective preliminary screening or validation tool for assessing N-glycosylation status. • Broad system compatibility: PNGase F and A enzymes are active in mammalian, plant, and microbial systems, allowing reliable N-glycosylation assessment regardless of sample origin.