Pngase F
Pngase F - Related Videos
08:26
Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+
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Cited by 9 •
2018
We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).
11:27
Analysis of SCAP N-glycosylation and Trafficking in Human Cells
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Cited by 17 •
2016
We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.
11:38
A Quantitative Glycomics and Proteomics Combined Purification Strategy
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Cited by 18 •
2016
A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.
08:52
Spatial Molecular Imaging of the Glycome Using Mass Spectrometry
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Cited by 1 •
2025
This protocol details the key steps to enable rigorous and reproducible measurement of both glycogen and N-linked glycans using mass spectrometry imaging.
06:11
Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence
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Cited by 4 •
2023
Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.
FAQs
Beyond releasing the carbohydrate, the reaction changes the modified residue at the glycosylation site: asparagine becomes aspartic acid. This conversion provides a defined molecular consequence of N-linked glycan removal, which is important when interpreting glycoprotein characterization results and distinguishing carbohydrate-dependent effects from changes associated with the polypeptide itself.
Cleavage depends on the structural arrangement of the N-linked glycan near the innermost N-acetylglucosamine and asparagine bond. Certain core fucose linkages can prevent the enzyme from cutting that bond. Consequently, treatment may remove many N-linked glycans while leaving particular structures attached, so incomplete deglycosylation should be considered when interpreting the resulting molecular pattern.
Carbohydrate chains can contribute to complex patterns in glycoprotein analyses. Removing the N-linked portion simplifies those patterns, making changes in the protein-related signal easier to examine. Comparing the glycoprotein before and after treatment can therefore support investigations of molecular weight, structural features, processing, and the contribution of glycosylation to the observed result.
A typical analytical workflow uses PNGase F treatment as a preparation step before examining the glycoprotein by molecular-weight analysis, electrophoresis, immunoblotting, or mass spectrometry. The enzyme reduces complexity caused by N-linked carbohydrates, allowing subsequent measurements to focus more clearly on the protein-associated pattern and on differences attributable to glycosylation.
Deglycosylation can simplify the patterns observed during electrophoresis and immunoblotting by removing N-linked carbohydrate contributions from the analyzed glycoprotein. This makes those methods useful for examining molecular-weight differences and comparing glycosylated material with material after treatment. Residual signal complexity may remain when the sample contains structures that the enzyme does not cleave.
Mass spectrometry can be easier to interpret when N-linked carbohydrate complexity has been reduced. PNGase F treatment releases susceptible oligosaccharides and converts the associated asparagine residue to aspartic acid, producing a chemically defined treated form. The resulting data can support glycoprotein characterization while preserving awareness that certain core fucose-containing structures may resist cleavage.