PNGase F
Peptide-N-Glycosidase F(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 inner most GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins.
Application
This enzyme is useful for removal of carbohydrate residues from proteins.
Preparation and specification
|
Appearance |
Colorless Liquid |
|
Protein purity |
≥95% (from SDS-PAGE) |
|
Activity |
≥500,000 U/mL |
|
Exoglycosidase |
No activity could be detected (ND) |
|
Endoglycosidase F1 |
ND |
|
Endoglycosidase F2 |
ND |
|
Endoglycosidase F3 |
ND |
|
Endoglycosidase H |
ND |
|
Protease |
ND |
Properties
|
EC number |
3.5.1.52 (Recombinant from microorganism) |
|
Molecular weight |
35 kDa (SDS-PAGE) |
|
Isoelectric point |
8. 14 |
|
Optimum pH |
7.0-8.0 |
|
Optimum temperature |
65 °C |
|
Substrate specificity |
Cleaving glycosidic bonds between GlcNAc and asparagine residues Fig.1 |
|
Recognition sites |
N-linked glycans unless containing α1-3 fucose Fig. 2 |
|
Activators |
DTT |
|
Inhibitor |
SDS |
|
Storage temperature |
-25 ~-15 ℃ |
|
Heat Inactivation |
A 20µL reaction mixture containing 1µL of PNGase F is inactivated by incubation at 75 °C for 10 minutes. |
Fig. 1 Substrate specificity 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.
Components
|
|
Components |
Concentration |
|
1 |
PNGase F |
50 µl |
|
2 |
10×Glycoprotein Denaturing Buffer |
1000 µl |
|
3 |
10×GlycoBuffer 2 |
1000 µl |
|
4 |
10% NP-40 |
1000 µl |
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.
