Peptide N-glycosidase F (fast version)
PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins.
PREPARATION and SPECIFICATION
This enzyme is useful for removal of carbohydrate residues from proteins.
Fig. 2 Recognition sits of PNGase F. When the internal GlcNAc residues are linked to α1-3 fucose, PNGase F cannot cleave N-linked oligosaccharides from glycoproteins. This modification is common in plants and some insect glycoproteins.
One unit(U) is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37 °C in a total reaction volume of 10 µL.
- Dissolve 1-20 µg of glycoprotein with deionized water, add 1 µl 10×Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume.
- Incubate at 100 °C for 10 min, cool it on ice.
- Add 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 and mix.
- Add 1-2 µl PNGase F and H2O (if necessary) to make a 20 µl total reaction volume and mix.
- Incubate reaction at 37 °C for 60 min.
- For SDS-PAGE analysis or HPLC analysis.
- Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
- Tretter, V. et al. (1991). Eur. J. Biochem. 199, 647-652.
- Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.