| Species : |
Flavobacterium meningosepticum |
| Description : |
PNGase F is the most effective enzymatic method for removing nearly all N-linked oligosaccharides from glycoproteins. As an amidase, PNGase F can cleave between the innermost GlcNAc and asparagine residues in high-mannose, hybrid, and complex-type oligosaccharides. |
| Molecular Mass : |
36 kDa |
| Unit Definition : |
One unit is defined as the amount of enzyme required to remove over 95% of the glycan chains from 10 μg of denatured RNase B in a total reaction volume of 10 μL at 37 centigrade for 1 hour. |
| Unit Definition Assay : |
Ten micrograms of RNase B (control substrate) is denatured for 10 minutes at 100 centigrade using 1× Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT). After the addition of NP-40 and GlycoBuffer 2, two-fold diluted PNGase F is added, and the reaction mixture is incubated at 37 centigrade for 1 hour. The reaction products are then separated and observed by SDS-PAGE.
1× Glycoprotein Denaturing Buffer: 0.5% SDS, 40 mM DTT
1× NP-40: 1% NP-40 (dissolved in MilliQ-H2O) |
| Purity : |
≥ 95% (determined by SDS-PAGE and ESI-MS) |
| Heat Inactivation : |
75 centigrade for 10 minutes |
| Note : |
1. SDS inhibits the activity of PNGase F, so the reaction mixture must contain NP-40. The mechanism by which this nonionic detergent counteracts the inhibitory effect of SDS is not yet clear.
2. To deglycosylate glycoproteins under non-denaturing conditions, it is recommended to increase the enzyme amount and extend the incubation time.
3. PNGase F does not cleave N-linked glycans containing a core α1-3 fucose. |
| Storage Buffer : |
20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50% Glycerol, pH 7.5 at 25 centigrade. |
| 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. |
