PNGase F

PNGase F (Peptide N-Glycosidase F) is derived from the gene of Elizabethkingia miricola and is expressed in E. coli, with a molecular weight of 35 kDa. PNGase F is an amidase that cleaves N-linked glycans from glycoproteins. It acts upon high-mannose, hybrid, and complex oligosaccharide types. The cleavage site is the glycosidic bond between the innermost N-acetylglucosamine residue and the asparagine residue; it is capable of releasing the vast majority of N-linked glycans (with the exception of those containing a core α-1,3-linked fucose). This enzyme is suitable for the efficient release of N-linked oligosaccharides from glycoproteins in proteomics and glycobiology research. The buffer composition consists of 20 mM Tris-HCl, 200 mM NaCl (pH 7.5), and 10% glycerol.
1. Deglycosylation of Glycoproteins under Denaturing Conditions
1) Dissolve 1-20 µg of glycoprotein in deionized water. Add 1 µL of 10× Glycoprotein Denaturing Buffer, and bring the final volume to 10 µL using deionized water.
2) Incubate at 75°C for 10 minutes.
3) Add 2 µL of 10× GlycoBuffer and 2 µL of 10% NP-40; gently pipette to mix.
4) Add 1-2 µL of PNGase F, bring the final volume to 20 µL using deionized water, and gently pipette to mix.
5) Incubate at 37°C for 1–3 hours.
6) Proceed with SDS-PAGE or HPLC analysis.
2. Glycoprotein Deglycosylation under Non-denaturing Conditions
1) Take 10-100 µg of glycoprotein solution, add 2 µL of 10×Glyco buffer, 2-5 µL of PNGase F (0.1-1 μg), and add purified water to make the total volume of the reaction system 20 µL. Mix gently.
2) Incubate at 37°C for 4-24 hours.
Note: When deglycosylating native glycoproteins, it is recommended to prepare an equal amount of the glycoprotein sample under denaturing conditions and perform a simultaneous enzymatic digestion as a positive control. This allows for the determination of the extent of the deglycosylation reaction under non-denaturing conditions.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.