Peptide N-glycosidase F (fast version)

PRODUCT DESCRIPTION

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

PROPERTIES

APPLICATIONS

This enzyme is useful for removal of carbohydrate residues from proteins.

Recognition sits of PNGase F

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.

REAGENT COMPOSITION

UNIT DEFINITION

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.

REACTION CONDITIONS

  1. 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.
  2. Incubate at 100 °C for 10 min, cool it on ice.
  3. Add 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 and mix.
  4. Add 1-2 µl PNGase F and H2O (if necessary) to make a 20 µl total reaction volume and mix.
  5. Incubate reaction at 37 °C for 60 min.
  6. For SDS-PAGE analysis or HPLC analysis.

REFERENCE

  1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
  2. Tretter, V. et al. (1991). Eur. J. Biochem. 199, 647-652.
  3. Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.