PNGase F-Fast
PNGase Fa 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. It is very important for effective process control to obtain the accurate distribution of N-glycans in a short time. Generally, it takes several hours to hydrolyze and release the N-glycans of the antibody with PNGase F, and then carry out glycan derivatization and HPLC or mass spectrometry analysis. Especially, incomplete deglycosylation can lead to biased results, which will not represent the correct composition of the therapeutic antibody. PNGase F-Fast is an improved reagent, which can release the N-glycans of therapeutic antibody in a few minutes. All N-glycans can be released quickly without bias, and are ready for downstream chromatography or mass spectrometry analysis, so as to determine the glycoprofile of antibodies quickly.
Application
This enzyme is useful for removal of carbo hydrate residues from antibodies and fusion proteins in only minutes.
Preparation and specification
|
Appearance |
Colorless Liquid |
|
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 20 µL reaction mixture containing 1 µL of PNGase F is inactivated by incubation at 75 °C for 10 minutes. |
Fig. 1 Substrate specificity of PNGase F-Fast
Fig. 2 Cleavage sits of IgG. The red arrow indicates the Cleavage sites of IgG.
Components
|
Components |
Volume |
|
PNGase F-Fast |
50 µl |
|
5×PNGase F-Fast Buffer |
1000 µl |
Reaction conditions
One-step Protocol:
1. Dissolve 100 µg of antibody with deionized water to make a volume of 16 µl.
2. Add 4 µl 5×PNGase F-Fast buffer and mix.
3. Add 1 µl PNGase F-Fast and mix.
4. Incubate reaction at 50 °C for 10 min.
5. Prepare N-glycans for derivatization for downstream analysis.
Two-step Protocol:
Some antibodies require a preheating step for efficient deglycosylation.
1. Dissolve 100 µg of antibody with deionized water to make a volume of 16 µl.
2. Add 4 µl 5×PNGase F-Fast buffer and mix.
3. Incubate at 80 °C for 2 min and cool on ice.
4. Add 1 µl PNGase F-Fast and mix.
5. Incubate reaction at 50 °C for 10 min.
6. Prepare N-glycans for derivatization for downstream analysis.
